Specific inhibitors of NFAT activation by calcineurin and their use in treating immune-related diseases

ABSTRACT

Isolated peptide fragments of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof are described. Isolated polynucleotides and gene therapy vectors encoding such peptide fragments are also described. In addition, methods for treating immune-related diseases or conditions and methods for high throughput screening of candidate agents are described. Pharmaceutical compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 10/066,151, filed on Jan. 31, 2002, which is acontinuation of U.S. patent application Ser. No. 09/248,620, filed Feb.11, 1999, which claims the benefit of U.S. Provisional Application No.60/074,467, filed Feb. 12, 1998, all of which are incorporated herein byreference in their entirety.

[0002] This invention was made with Government support under NationalInstitutes of Health Grant Nos. AI 40127, AI 43726, GM 38608, and GM47467. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention relates generally to NFAT peptide fragments, NFATpolynucleotides, NFAT gene therapy vectors, treatments for immune systemrelated diseases, and methods for identifying immunosuppressive agents.

BACKGROUND OF THE INVENTION

[0004] Hyperactivity or inappropriate activity of the immune system is aserious and widespread medical problem. It contributes to acute andchronic immune diseases, e.g., allergic and atopic diseases, e.g.,asthma, allergic rhinitis, allergic conjunctivitis and atopicdermatitis, and to autoimmune diseases, e.g., rheumatoid arthritis,insulin-dependent diabetes, inflammatory bowel disease, autoimmunethyroiditis, hemolytic anemia and multiple sclerosis. Hyperactivity orinappropriate activity of the immune system is also involved intransplant graft rejections and graft-versus-host disease.

[0005] A certain family of transcription factors, the NFAT proteins, areexpressed in immune cells and play a key role in eliciting immuneresponses. The NFAT proteins are activated by calcineurin, and theactivated NFAT proteins, in turn, induce transcription of cytokine geneswhich are required for an immune response. The immunosuppressive drugscyclosporin A and FK506 are potent inhibitors of cytokine genetranscription in activated immune cells, and have been reported to actby inhibiting calcineurin such that calcineurin is not able to activateNFAT. These drugs, however, can display nephrotoxic and neurotoxiceffects after long term usage. Since calcineurin is ubiquitouslyexpressed in many tissues, the drugs' inhibition of calcineurin activitytoward substrates other than NFAT may contribute to the observedtoxicity.

[0006] There is a need for immunosuppressive agents which selectivelyinhibit the calcineurin-NFAT interactions and which do not inhibit theenzymatic activity of calcineurin for its other substrates.

SUMMARY OF THE INVENTION

[0007] It is an object of the invention to provide an immunosuppressiveagent with reduced toxic effects.

[0008] It is another object of the invention to provide animmunosuppressive agent that inhibits interaction between calcineurinand NFAT.

[0009] It is yet another object of the invention to provide animmunosuppressive agent that selectively inhibits interaction betweencalcineurin and NFAT, and which does not inhibit enzymatic activity ofcalcineurin for its other substrates.

[0010] It is yet another object of the invention to provide a genetherapy vector encoding an immunosuppressive agent that selectivelyinhibits interaction between calcineurin and NFAT.

[0011] It is yet another object of the invention to provide a method forinhibiting an immune response using an immunosuppressive agent thatselectively inhibits interaction between calcineurin and NFAT.

[0012] It is yet another object of the invention to provide methods forhigh-throughput screening of candidate agents to identify agents thatinhibit one or more aspects of calcineurin-mediated NFAT activation.

[0013] According to the invention, an isolated fragment of the conservedregulatory domain of NFAT protein, e.g., NFAT1, NFAT2, NFAT3 or NFAT4,capable of inhibiting protein-protein interaction between calcineurinand NFAT, or a biologically active analog thereof, is provided.Preferably, the peptide fragment or biologically active analog thereofdoes not inhibit or does not substantially inhibit the activity ofcalcineurin toward non-NFAT calcineurin substrates.

[0014] In certain embodiments, the peptide fragment comprises the aminoacid sequence IX₂X₃T (SEQ ID NO:104), wherein X₂ is E, R or Q, and X₃ isI or F. Preferred amino acid sequences are, e.g., IEIT (SEQ ID NO:105),IRIT (SEQ ID NO:106), IQIT (SEQ ID NO:107), and IQFT (SEQ ID NO:108).

[0015] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₁IX₂X₃T (SEQ ID NO:73), wherein X₁ is R or S, X₂ is E, Ror Q, and X₃ is I or F. Preferred amino acid sequences are, e.g.,X₁IX₂IT (SEQ ID NO:74), RIX₂IT (SEQ ID NO:75), X₁IEIT (SEQ ID NO:76),RIEIT (SEQ ID NO:1), SIRIT (SEQ ID NO:2), SIQIT (SEQ ID NO:3), and SIQFT(SEQ ID NO:4).

[0016] In certain embodiments, the peptide fragment comprises the aminoacid sequence PX₁IX₂X₃T (SEQ ID NO:77), wherein X₁ is R or S, X₂ is E, Ror Q, and X₃ is I or F. Preferred amino acid sequences are, e.g., PRIEIT(SEQ ID NO:5), PSIRIT (SEQ ID NO:6), PSIQIT (SEQ ID NO:71) and PSIQFT(SEQ ID NO:7).

[0017] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₅PX₁IX₂X₃T (SEQ ID NO:78), wherein X₁ is R or S, X₂ is E,R or Q, X₃ is I or F and X₅ is S or C. Preferred amino acid sequencesare, e.g., SPRIEIT (SEQ ID NO:8), CPSIRIT (SEQ ID NO:9), CPSIQIT (SEQ IDNO:10) and CPSIQFT (SEQ ID NO:11).

[0018] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₅PX₁IX₂X₃TX₆ (SEQ ID NO:79), wherein X₁ is R or S, X₂ isE, R or Q, X₃ is I or F, X₅ is S or C, and X₆ is P or S. Preferred aminoacid sequences are, e.g., SPRIEITP (SEQ ID NO:12), SPRIEITS (SEQ IDNO:13), CPSIRITS (SEQ ID NO:14), CPSIQITS (SEQ ID NO:15) and CPSIQFTS(SEQ ID NO:16).

[0019] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₅PX₁IX₂X₃TX₆X₇ (SEQ ID NO:80), wherein X₁ is R or S, X₂is E, R or Q, X₃ is I or F, X₅ is S or C, X₆ is P or S, and X₇ is S, Cor I. Preferred amino acid sequences are SPRIEITPS, (SEQ ID NO:17)SPRIEITSC, (SEQ ID NO:18) CPSIRITSI, SEQ ID NO:19) CPSIQITSI, and (SEQID NO:20) CPSIQFTSI. (SEQ ID NO:21)

[0020] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₁₁X₁₀X₉X₅PX₁IX₂X₃TX₆X₇X₈ (SEQ ID NO:81), wherein X₁ is Ror S, X₂ is E, R or Q, X₃ is I or F, X₅ is S or C, X₆ is P or S, X₇ isS, C or I, X₈ is H, L or S, X₉ is P, L or E, X₁₀, is G, L or F, and X₁₁is S, A, V or P. Preferred amino acid sequences are, e.g., SGPSPRIEITPSH(SEQ ID NO:22), SGLSPRIEITPSH (SEQ ID NO:23), ALESPRIEITSCL (SEQ IDNO:24), VLECPSIRITSIS (SEQ ID NO:25), PFECPSIQITSIS (SEQ ID NO:26),PFECPSIQITSIS (SEQ ID NO:27) and PFECPSIQFTSIS (SEQ ID NO:28). Otherpreferred amino acid sequences are, e.g., KPAGASGPSPRIEITPSHELMQAGG,(SEQ ID NO:29) KPAGASGLSPRIEITPSHELIQAVG, (SEQ ID NO:30)PDGAPALESPRIEITSCLGLYHNNN, (SEQ ID NO:31) AGGGRVLECPSIRITSISPTPEPPA,(SED ID NO:32) LGGPKPFECPSIQITSISPNCHQEL, (SEQ ID NO:33)LGGPKPFECPSIQITSISPNCHQGT and (SEQ ID NO:34) LGGPKPFECPSIQFTSISPNCQQEL.(SEQ ID NO:35)

[0021] Another aspect of the invention is an isolated polynucleotideencoding the peptide comprising the amino acid sequence as set forth inSEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, orbiologically active analogs thereof. Preferred polynucleotide sequencesare, e.g., the sequences as set forth in SEQ ID NO:110, SEQ ID NO:111,SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115.

[0022] Another aspect of the invention is an isolated polynucleotideencoding the peptide comprising the amino acid sequence as set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or biologicallyactive analogs thereof. Preferred polynucleotide sequences are, e.g.,the sequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:83 and SEQ ID NO:84.

[0023] Another aspect of the invention is an isolated polynucleotideencoding the peptide comprising the amino acid sequence as set forth inSEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:71, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, or biologically active analogsthereof. Preferred polynucleotide sequences are, e.g., the sequences asset forth in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42, SEQID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85, SEQ IDNO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91 and SEQ ID NO:92.

[0024] Another aspect of the invention is an isolated polynucleotideencoding the peptide comprising the amino acid sequence as set forth inSEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, or biologically active analogs thereof.Preferred polynucleotide sequences are, e.g., the sequences as set forthin SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62 and SEQ ID NO:63.

[0025] Another aspect of the invention is an isolated polynucleotideencoding the peptide comprising the amino acid sequence as set forth inSEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, or biologically active analogs thereof.Preferred polynucleotide sequences are, e.g., sequences as set forth inSEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69 and SEQ ID NO:70.

[0026] Another aspect of the invention is a gene therapy vectorcomprising a nucleotide sequence encoding a peptide fragment of theconserved regulatory domain of NFAT protein capable of inhibitingprotein-protein interaction between calcineurin and NFAT, or abiologically active analog of the peptide fragment.

[0027] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107,SEQ ID NO:108, or biologically active analogs thereof. In certainembodiments, the gene therapy vector comprises the nucleotide sequencesas set forth in SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ IDNO:113, SEQ ID NO:114 or SEQ ID NO:115.

[0028] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, or biologically active analogs thereof. In certain embodiments,the gene therapy vector comprises the nucleotide sequences as set forthin SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:83or SEQ ID NO:84.

[0029] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:71, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, orbiologically active analogs thereof. In certain embodiments, the genetherapy vector comprises the nucleotide sequence as set forth in SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ IDNO:92.

[0030] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or biologicallyactive analogs thereof. In certain embodiments, the gene therapy vectorcomprises the nucleotide sequence as set forth in SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ IDNO:63.

[0031] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, or biologicallyactive analogs thereof. In certain embodiments, the gene therapy vectorcomprises the nucleotide sequence as set forth in SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 or SEQ IDNO:70.

[0032] Another aspect of the invention is a cell having a gene therapyvector described herein.

[0033] Another aspect of the invention is a method for producing apeptide capable of inhibiting protein-protein interaction betweencalcineurin and NFAT, comprising culturing a cell having a gene therapyvector described herein under conditions that permit expression of thepeptide.

[0034] Another aspect of the invention is a method for treating animmune-related disease or condition, e.g., acute immune diseases,chronic immune diseases or autoimmune diseases, in an animal. It is alsomeant to include treatment of tissue or organ transplant graftrejections or graft-versus-host disease. A gene therapy vector describedherein is administered to the animal.

[0035] Another aspect of the invention is a method for providing ananimal having an immune-related disease or condition with atherapeutically effective level of a peptide capable of inhibitingprotein-protein interaction between calcineurin and NFAT. A gene therapyvector described herein is administered to the animal.

[0036] Another aspect of the invention is a method for inhibiting animmune response in an animal. An animal in need of inhibition of animmune response is provided. A therapeutically effective amount of apeptide fragment of the conserved regulatory domain of NFAT proteincapable of inhibiting protein-protein interaction between calcineurinand NFAT, or a biologically active analog thereof, is provided. Thepeptide fragment or biologically active analog thereof is administeredto the animal so as to inhibit the immune-response in the animal.

[0037] In certain embodiments, the therapeutically effective amount ofthe peptide fragment is provided by providing to the animal arecombinant nucleic acid having a nucleotide sequence encoding thepeptide fragment or a biologically active analog thereof, and which iscapable of expressing the peptide fragment or biologically active analogthereof in vivo. The peptide fragment is administered to the animal byadministering the recombinant nucleic acid. The nucleic acid can be,e.g., any of the polynucleotides described herein.

[0038] In certain embodiments, the therapeutically effective amount ofthe peptide fragment is provided by providing to the animal acomposition comprising animal cells wherein a recombinant nucleic acidhaving a nucleotide sequence encoding the peptide fragment has beenintroduced ex vivo into the animal cells so as to express the peptidefragment in the animal cells. The peptide fragment is administered tothe animal by administering the animal cells having the recombinantnucleic acid. Preferably, the recombinant nucleic acid is a gene therapyvector, e.g., as described herein. Preferably, the animal cells arederived from the animal to be treated or allogeneic cells.

[0039] Another aspect of the invention is a method for treating adisease involving hyperactivity or inappropriate activity of the immunesystem, a transplant graft rejection or graft-versus-host disease, in ananimal. An animal in need of treatment for a disease involvinghyperactivity or inappropriate activity of the immune system, atransplant graft rejection or graft-versus-host disease, is provided. Atherapeutically effective amount of a peptide fragment of the conservedregulatory domain of NFAT protein capable of inhibiting protein-proteininteraction between calcineurin and NFAT, or a biologically activeanalog thereof, is provided. The peptide fragment or biologically activeanalog thereof is administered to the animal in a therapeuticallyeffective amount such that treatment of the disease involvinghyperactivity or inappropriate activity of the immune system, transplantgraft rejection or graft-versus-host disease, occurs.

[0040] Another aspect of the invention is a method for treating ananimal at risk for a disease involving hyperactivity or inappropriateactivity of the immune system, a transplant graft rejection orgraft-versus-host disease. An animal at risk for a disease involvinghyperactivity or inappropriate activity of the immune system, atransplant graft rejection or graft-versus-host disease, is provided. Atherapeutically effective amount of a peptide fragment of the conservedregulatory domain of NFAT protein capable of inhibiting protein-proteininteraction between calcineurin and NFAT, or a biologically activeanalog thereof, is provided. The peptide fragment or biologically activeanalog thereof is administered in a therapeutically effective amountsuch that treatment occurs.

[0041] Another aspect of the invention is a method for gene therapy. Ananimal cell is genetically modified such that it is able to express apeptide fragment or biologically active analog thereof of the conservedregulatory domain of NFAT protein, the peptide fragment being capable ofinhibiting calcineurin-mediated NFAT activation, so as to effect genetherapy. In certain embodiments, the animal cells are geneticallymodified by introducing into the cells a recombinant nucleic acid havinga nucleotide sequence encoding the peptide fragment and which is capableof expressing the peptide fragment in vivo. Preferably, the recombinantnucleic acid is a gene therapy vector, e.g., as described herein.

[0042] Another aspect of the invention is a pharmaceutical compositionfor treating an immune-related disease or condition in an animalcomprising a therapeutically effective amount of a peptide fragment ofthe conserved regulatory domain of NFAT protein capable of inhibitingprotein-protein interaction between calcineurin and NFAT, or abiologically active analog thereof, and a pharmaceutically acceptablecarrier. The peptide fragment can be, e.g., any of the peptide fragmentsdescribed herein.

[0043] Another aspect of the invention is a pharmaceutical compositionfor treating an immune-related disease or condition in an animal,comprising a therapeutically effective amount of a recombinant nucleicacid having a nucleotide sequence encoding a peptide fragment of theconserved regulatory domain of NFAT protein capable of inhibitingprotein-protein interaction between calcineurin and NFAT, or abiologically active analog thereof, and a pharmaceutically acceptablecarrier. The nucleic acid can be, e.g., any of the polynucleotidesdescribed herein.

[0044] Another aspect of the invention is a pharmaceutical compositionfor treating an immune-related disease or condition in an animal,comprising a therapeutically effective amount of animal cells wherein arecombinant nucleic acid having a nucleotide sequence encoding a peptidefragment of the conserved regulatory domain of NFAT protein capable ofinhibiting protein-protein interaction between calcineurin and NFAT, ora biologically active analog thereof, has been introduced into theanimal cells so as to express the peptide fragment; and apharmaceutically acceptable carrier. Preferably, the animal cells arecells derived from the animal to be treated or allogeneic cells.

[0045] Another aspect of the invention is a method for inhibitingprotein-protein interaction between calcineurin and NFAT in vivo. A cellhaving calcineurin and NFAT is provided. A peptide fragment or abiologically active analog thereof of the conserved regulatory domain ofNFAT protein capable of inhibiting protein-protein interaction betweencalcineurin and NFAT is provided. The calcineurin and peptide fragmentor biologically active analog thereof are contacted in vivo such thatprotein-protein interaction between the calcineurin and the NFAT isinhibited.

[0046] Another aspect of the invention is a method for inhibitingprotein-protein interaction between calcineurin and NFAT in vitro.Calcineurin and NFAT are provided. A peptide fragment or a biologicallyactive analog thereof of the conserved regulatory domain of NFAT proteincapable of inhibiting protein-protein interaction between calcineurinand NFAT is provided. The calcineurin and peptide fragment orbiologically active analog thereof are contacted in vitro such thatprotein-protein interaction between the calcineurin and the NFAT isinhibited.

[0047] Another aspect of the invention is a method for evaluating anagent for use in modulating an immune response. A cell is provided. Anagent, e.g., a peptide fragment of the conserved regulatory domain ofNFAT protein or biologically active analogs thereof, is provided. Theeffect of the agent on an aspect of calcineurin-mediated NFAT activationis evaluated, e.g., protein-protein interaction between calcineurin andNFAT, dephosphorylation of NFAT by calcineurin, recruitment of NFAT tothe nucleus in a cell, conformational change in NFAT, or activation ofNFAT-dependent gene transcription. A change in the aspect ofcalcineurin-mediated NFAT activation is indicative of the usefulness ofthe agent in modulating an immune response.

[0048] Another aspect of the invention is a method for high throughputscreening of candidate agents to identify an agent that inhibitsprotein-protein interaction between calcineurin and NFAT. A firstcompound is provided. The first compound is calcineurin or abiologically active derivative thereof, or the first compound is NFAT ora biologically active derivative thereof. A second compound is providedwhich is different from the first compound and which is labeled. Thesecond compound is calcineurin or a biologically active derivativethereof, or the second compound is NFAT or a biologically activederivative thereof. A candidate agent is provided. The first compound,second compound and candidate agent are contacted with each other. Theamount of label bound to the first compound is determined. A reductionin protein-protein interaction between the first compound and the secondcompound as assessed by label bound is indicative of the usefulness ofthe agent in inhibiting protein-protein interaction between calcineurinand NFAT. In certain embodiments, the method includes a washing stepafter the contacting step, so as to separate bound and unbound label.

[0049] Another aspect of the invention is a method for high throughputscreening of candidate agents to identify an agent that inhibitsprotein-protein interaction between calcineurin and NFAT. A firstcompound is provided. The first compound is calcineurin or abiologically active active derivative thereof, or the first compound isan organic molecule capable of binding to calcineurin and inhibitingprotein-protein interaction between calcineurin and NFAT. A secondcompound is provided which is different from the first compound andwhich is labeled. The second compound is calcineurin or a biologicallyactive active derivative thereof, or the second compound is an organicmolecule capable of binding to calcineurin and inhibitingprotein-protein interaction between calcineurin and NFAT. A candidateagent is provided. The first compound, second compound and candidateagent are contacted with each other. The amount of label bound to thefirst compound is determined. A reduction in protein-protein interactionbetween the first compound and the second compound as assessed by labelbound is indicative of the usefulness of the agent in inhibitingprotein-protein interaction between calcineurin and NFAT. In certainembodiments, the method includes a washing step after the contactingstep, so as to separate bound and unbound label.

[0050] In another aspect, the invention provides a method for highthroughput screening of candidate agents to identify an agent thatinhibits protein-protein interaction between calcineurin and NFAT. Afirst compound is provided. The first compound is NFAT or a biologicallyactive active derivative thereof, or the first compound is an organicmolecule capable of binding to NFAT and inhibiting protein-proteininteraction between calcineurin and NFAT. A second compound is providedwhich is different from the first compound and which is labeled. Thesecond compound is NFAT or a biologically active active derivativethereof, or the second compound is an organic molecule capable ofbinding to NFAT and inhibiting protein-protein interaction betweencalcineurin and NFAT. A candidate agent is provided. The first compound,second compound and candidate agent are contacted with each other. Theamount of label bound to the first compound is determined. A reductionin protein-protein interaction between the first compound and the secondcompound as assessed by label bound is indicative of the usefulness ofthe agent in inhibiting protein-protein interaction between calcineurinand NFAT. In certain embodiments, the method includes a washing stepafter the contacting step, so as to separate bound and unbound label.

[0051] Another aspect of the invention is a method for high-throughputscreening of candidate agents to identify an agent that inhibitsdephosphorylation of NFAT by calcineurin. Phosphorylated NFAT isprovided. Calcineurin or a biologically active derivative thereof havingenzymatic activity is provided. A candidate agent is provided. Thephosphorylated NFAT, the calcineurin or biologically active derivativethereof, and the candidate agent are contacted with each other inreaction media, e.g., buffer, under conditions that allow enzymaticactivity of calcineurin. In certain embodiment, the NFAT is separatedfrom the reaction media. It is determined whether phosphate remainsassociated with the NFAT. If phosphate remains associated with NFAT, itis indicative of the usefulness of the agent in inhibitingdephosphorylation of NFAT by calcineurin.

[0052] Another aspect of the invention is a method for high-throughputscreening of candidate agents to identify an agent that inhibitsconformational change in NFAT from dephosphorylation by calcineurin.Phosphorylated NFAT is provided. Calcineurin or a biologically activederivative thereof having enzymatic activity is provided. A candidateagent is provided. An oligonucleotide having an NFAT site is provided.The phosphorylated NFAT, calcineurin or biologically active derivativethereof, and the candidate agent are contacted with each other inreaction media under conditions that allow enzymatic activity ofcalcineurin. Specific binding of NFAT to the oligonucletide having theNFAT site is determined. A reduction of binding is indicative of theusefulness of the agent in inhibiting conformational change in NFAT fromdephosphorylation by calcineurin.

[0053] Another aspect of the invention is a method for high-throughputscreening of candidate agents to identify an agent that inhibitscalcineurin-dependent import of NFAT into the nucleus of a cell. Cellsexpressing NFAT are provided. A stimulant that activates NFAT throughthe calcium/calcineurin pathway is provided. A candidate agent isprovided. The cells, stimulant and candidate agent are contacted witheach other. The presence or absence of nuclear NFAT in the cells isdetermined. A reduction in nuclear NFAT is indicative of the agentinhibiting calcineurin-dependent import of NFAT into the nucleus of acell.

[0054] Another aspect of the invention is a method for assessing thestate of NFAT activation of immune system cells from an animal. Immunesystem cells isolated from an animal are provided. The presence orabsence of nuclear NFAT in the cells is determined. The presence ofnuclear NFAT in the cells is indicative of activation of NFAT in thecells.

[0055] Another aspect of the invention is a method for assessing theability of immune system cells isolated from an animal to respond to anNFAT activating signal. Immune system cells from an animal are provided,the cells being unactivated for NFAT. A stimulant that activates NFAT isprovided. The cells are contacted with the stimulant. The presence orabsence of nuclear NFAT in the cells is determined. A reduction innuclear NFAT is indicative of impairment of the ability of the cells torespond to an NFAT activating signal.

[0056] Another aspect of the invention is a method for identifying astimulant that can activate NFAT in immune system cells isolated from ananimal. Immune system cells isolated from an animal are provided. Acandidate stimulant, e.g., allergen, is provided. The cells arecontacted with the candidate stimulant. The presence or absence ofnuclear NFAT in the cells is determined. The presence of nuclear NFAT isindicative of the stimulant activating NFAT in the cells.

[0057] Another aspect of the invention is a method for identifying anallergen. An animal cell line expressing NFAT is provided. IgE from ananimal, e.g., a human, is provided. A candidate allergen is provided.The cell line is contacted with the IgE. The cell line is contacted withthe candidate allergen. The presence or absence of nuclear NFAT in cellsof the cell line is determined. The presence of nuclear NFAT isindicative of the candidate allergen being an allergen.

[0058] In another aspect, the invention features a method for treatingor preventing an NFAT-related disease or disorder, e.g., a disease ordisorder caused by excessive or inappropriate activation of NFAT or amolecular target of NFAT, e.g., an immune disease, a cardiovasculardisease, a skeletal muscle disease, a neurological disorder, or a weightdisorder, in an animal. The method includes administering to an animalan agent, e.g., an organic molecule (e.g., an an organic moleculedescribed herein), that modulates, e.g., inhibits, an interaction (e.g.,a protein-protein interaction, e.g., binding) between calcineurin andNFAT, in an amount sufficient to treat the NFAT-related disease ordisorder in the animal.

[0059] In another aspect, the invention features a pharmaceuticalcomposition comprising a therapeutically effective amount of an organicmolecule capable of inhibiting protein-protein interaction betweencalcineurin and NFAT, and a pharmaceutically acceptable carrier. Theagent can be, e.g., an agent that inhibits dephosphorylation of NFAT bycalcineurin. The molecular weight of the organic molecule can be lessthan 2500 Da, e.g., about 100 to 2000 Da, about 200 to 1500 Da, or about300 and 1000 Da. The organic molecule can bind to calcineurin with anaffinity constant of at least about 2×10⁴ M⁻¹, e.g., with an affinityconstant of at least about 10⁶ M⁻¹, at least about 10⁷ M⁻¹, or at leastabout 10⁸ M⁻¹. The organic molecule can be a compound generallyrepresented by:

[0060] formula (I):

[0061] wherein R¹ is hydrogen, C₁-C₂₀ alkyl optionally substituted with1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryloptionally substituted with 1-4 R⁶, heterocyclyl optionally substitutedwith 1-3 R⁶; heteroaryl optionally substituted with 1-4 R⁶; C₂-C₈alkenyl, or C₂-C₈ alkynyl, cyano, nitro, carboxy, carbo(C₁-C₆)alkoxy,trihalomethyl, halogen, C₁-C₆ alkoxy, hydroxy, aryloxy, acylamino,alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido; R² is C₁-C₂₀alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionallysubstituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶,heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionallysubstituted with 1-4 R⁶, C₁-C₆ alkoxy, or hydroxy; R³ is hydrogen orhalogen; R⁴ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionallysubstituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3R⁶, heteroaryl optionally substituted with 1-4 R⁶, or halogen; R⁵ isNR⁷, O or S; R⁶ is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl,alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino,dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;and R⁷ is C₁-C₆ alkyl;

[0062] formula (II):

[0063] wherein: R¹ and R² are each independently hydrogen, halogen,amino, C₁-C₆alkylamino, di(C₁-C₆)alkylamino, arylamino, diarylamino, or4,4-dimethyl-2,6-dioxocyclohexyl; R³ is NR¹¹ or O; R⁴, R⁵ and R⁸ areeach independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro,haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino,dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;R⁶ is hydrogen, halogen, or when taken together with R⁷ forms a doublebond between the carbon atoms to which they are attached, R⁷ ishydrogen, halogen, or when taken together with R⁶ forms a double bondbetween the carbon atoms to which they are attached, R⁹ is OR³, or whentaken together with R¹⁰ forms a double bond between the carbon andnitrogen atoms to which they are attached, R¹⁰ is hydrogen, or whentaken together with R⁹ forms a double bond between the carbon andnitrogen atoms to which they are attached;

[0064] R¹¹ is SO₂R¹²; and R¹² is aryl optionally substituted with alkyl,R¹³ is alkyl or aryl; and

[0065] formula (III):

[0066] wherein R¹ and R⁴ are each independently O or NR⁸; R² and R³ areeach independently hydrogen, halogen, or R² and R³ together combine toform aryl optionally substituted with 1-4 R⁹; R⁵ is hydrogen, halogen,carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, orcyano; R⁶, R⁷ and R⁹ are each independently hydrogen, C₁-C₆ alkyl,halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy,aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino,acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano,mercapto or ureido; R⁸ is SO₂R¹⁰; and R¹⁰ is aryl optionally substitutedwith alkyl.

[0067] In another aspect, the invention features a method for inhibitingprotein-protein interaction between calcineurin and NFAT. The methodincludes providing calcineurin and NFAT, providing a pharmaceuticalcomposition, e.g., a pharmaceutical composition described above, andcontacting the calcineurin, NFAT, and pharmaceutical compositiontogether, such that the protein-protein interaction between calcineurinand NFAT is inhibited.

[0068] In another aspect, the invention features a method of inhibitingan immune response in an animal, which includes administering to theanimal an organic molecule, e.g., an organic molecule described herein,capable of inhibiting protein-protein interaction between calcineurinand NFAT. The organic molecule can be administered to the animal as apharmaceutical composition as described herein.

[0069] In another aspect, the invention features a method for treating adisease involving hyperactivity or inappropriate activity of the immunesystem. The method includes identifying an animal suffering from adisease involving hyperactivity or inappropriate activity of the immunesystem, and administering to the animal a therapeutically effectiveamount of a pharmaceutical composition, e.g., a pharmaceuticalcomposition comprising a therapeutically effective amount of an organicmolecule capable of inhibiting protein-protein interaction betweencalcineurin and NFAT, and a pharmaceutically acceptable carrier, e.g., apharmaceutical composition as described above, to thereby treat thedisease involving hyperactivity or inappropriate activity of the immunesystem. The disease involving hyperactivity or inappropriate activity ofthe immune system can be, e.g., an acute immune disease or disorder, achronic immune disease or disorder, or an autoimmune disease ordisorder.

[0070] In another aspect, the invention features a method for treating adisease involving excessive or inappropriate activation of NFAT, or amolecular target thereof. Molecular targets include, but are not limitedto genes involved in immune activation and inflammation, e.g., genesencoding cytokines and chemokines, e.g,. IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-8, IL-10, IL-13, GM-CSF, IFN-gamma, TNF-alpha,MIP-1alpha, MIP-1beta, RANTES, lymphotactin, IL-16, and IL-18, and cellsurface proteins, e.g., CD25, CD40L, CD69, CTLA-4, FasL, CD5, CD21,CD3-gamma, CD23, and LIGHT; transcription factors, e.g., NFkappaB p50;c-Rel; Oct2; NFAT2 (also called NFATc, NFATc1); Nur77; NF-IL3/E4BP4;EGR1; EGR2; and EGR3; and effectors and other targets, e.g., granzyme B;perforin; COX-2; CDK4; and p21/CIP1. Molecular targets also includegenes involved in immune anergy or tolerance, e.g., transcriptionalactivators and repressors, e.g., Jumonji, Ikaros, Groucho-relatedprotein-4, SATB1, NF-IL3/E4BP4, RNF19, HIF-1, Elf-1, and LAD/TSAd; genesencoding receptor and non-receptor tyrosine phosphatases, e.g.,RPTP-sigma, RPTP-kappa, and PTP-1B; genes encoding G-proteins or othersignalling proteins, enzymes, and cell surface receptors (e.g., Rab10;GBP-3; RGS-2; diacylglycerol kinase alpha; Mlp (MARCKS-like protein);LAD/TSAd; ZAP70; phosphoglycerate mutase; glutamate dehydrogenase;LDH-alpha; CD3 epsilon; 4-IBB ligand; CD98 heavy chain; FasL;cation-dependent mannose-6-phosphate receptor; gamma-aminobutyric acidreceptor-associated protein 1); and genes and proteins involved inproteolytic pathways, endocytosis, lysosomal degradation (e.g., SOCS-2;TRAF5; caspase 3; serpin 1b; and cystatin C); and others (e.g.,tetracycline transporter-like protein; M-CSF; osmotic stress protein 94;heme oxygenase 2a; calcyclin; CDK4. Molecular targets further includegenes involved in osteoclast differentiation and function, e.g.,tartrate-resistant acid phosphatase, osteoclast stimulating factor, andcalcitonin receptor; genes involved in cardiac hypertrophy, e.g., atrialnatriuretic factor, adenylosuccinate synthetase-1; and B-typenatriuretic peptide; and genes involved in viral replication andactivation, e.g., HIV-1 LTR, and gene(s) involved in calcium-dependentre-activation of latent KSHV; and other genes, e.g., IP3R; COX-2; eNOS;iNOS; CDK4; p21/CIP1; BMP2; Myf5; myosin heavy chain IIa; VEGF; andgene(s) involved in growth of vascular smooth muscle cells in responseto PDGF. The method includes identifying an animal suffering from adisease involving excessive or inappropriate activation of NFAT or amolecular target thereof; and administering to the animal atherapeutically effective amount of a pharmaceutical compositiondescribed herein, to thereby treat the disease involving excessive orinappropriate activation of NFAT or molecular target thereof.

[0071] In another aspect, the invention features a method ofmanufacturing an agent that inhibits protein-protein interaction betweencalcineurin and NFAT. The method includes providing an organic compoundcapable of inhibiting protein-protein interaction between calcineurinand NFAT, e.g., an organic compound as described herein, providing atleast one pharmaceutically acceptable carrier; and combining the organiccompound with the pharmaceutically acceptable carrier, to therebymanufacture an agent that inhibits protein-protein interaction betweencalcineurin and NFAT

[0072] The invention also features a method of manufacturing an agentthat inhibits protein-protein interaction between calcineurin and NFAT.The process includes carrying out a method to identify an agent thatinhibits protein-protein interaction between calcineurin and NFAT, e.g.,a method as described herein. In one embodiment, the method includesproviding a first compound selected from the group consisting ofcalcineurin or a biologically active derivative thereof, and NFAT or abiologically active derivative thereof; providing a second compoundselected from the group consisting of calcineurin or a biologicallyactive derivative thereof, and NFAT or a biologically active derivativethereof, wherein the second compound is different from the firstcompound, and wherein the second compound is labeled; providing acandidate agent; contacting the first compound, the second compound, andthe candidate agent with each other; and determining the amount of labelbound to the first compound, wherein a reduction in interaction betweenthe first compound and the second compound as assessed by label bound isindicative of usefulness of the candidate agent in inhibitingprotein-protein intereaction between calcineurin and NFAT; andmanufacturing the agent, to thereby make an agent that inhibitsprotein-protein interaction between calcineurin and NFAT. In oneembodiment, the first compound is calcineurin and the second compound isa biologically active derivative of NFAT. In another embodiment, thebiologically active derivative of NFAT comprises the amino acid sequenceGPHPVIVITGPHEE.

[0073] In some embodiments, the methods of manufacturing furthercomprise the step of manufacturing the agent into a form suitable foradministration to an animal via a particular route, e.g., an oral,parenteral, topical, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural, orintrasternal route.

[0074] In another aspect, the invention features a method for inhibitingprotein-protein interaction between calcineurin and NFAT. The methodincludes providing calcineurin and NFAT, providing an organic moleculecapable of inhibiting protein-protein interaction between calcineurinand NFAT, e.g., an organic compound described herein, and contacting thecalcineurin and the organic compound such that protein-proteininteraction between the calcineurin and the NFAT is inhibited.

[0075] In another aspect, the invention features a method oftransplanting an organ. The method includes: (a) providing an organ froma donor, (b) transplanting the organ into a recipient; and (c) before,during, and/or after (b), administering to the recipient an amount of anorganic molecule described herein capable of inhibiting protein-proteininteraction between calcineurin and NFAT, to thereby transplant anorgan. The organic molecule can be administered to the recipient in theform of a pharmaceutical composition as described herein. The term“donor” as used herein refers to an animal (human or non-human) fromwhom an organ or tissue can be obtained for the purposes oftransplantation to a recipient. The term “recipient” refers to an animal(human or non-human) into which an organ or tissue can be transferred.The term “organ(s)” is used throughout the specification as a generalterm to describe any anatomical part or member having a specificfunction in the animal. Further included within the meaning of this termare substantial portions of organs, e.g., cohesive tissues obtained froman organ. Such organs include but are not limited to kidney, liver,heart, intestine, e.g., large or small intestine, pancreas, and lungs.Further included in this definition are bones and blood vessels, e.g.aortic transplants.

[0076] The invention also features a method of treating an animal toprevent transplant rejection. The method includes (a) transplanting anorgan into an animal; and (b) before, during, or after (a),administering to the animal an organic compound, e.g., an organicmolecule desribed herein, in an amount sufficient to inhibit theprotein-protein interaction between calcineurin and NFAT, to therebyprevent transplant rejection in the animal. In an embodiment, theorganic compound can be administered as a pharmaceutical composition.

[0077] The above and other features, objects and advantages of thepresent invention will be better understood by a reading of thefollowing specification in conjunction with the drawings. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIGS. 1A-1C depict amino acid sequences of certainimmunosuppressive agents of this invention, including 5-mers, 6-mers,7-mers, 8-mers, 9-mers, 13-mers and 25-mers.

[0079] FIGS. 2A-2E depict cDNA sequences from murine NFAT1, human NFAT1,human NFAT2, human NFAT3, human NFAT4 and murine NFAT4, encoding certainimmunosuppressive agents of this invention, and the corresponding aminoacid sequences.

[0080] FIGS. 3A-3B depict the nucleotide and amino acid sequences of atruncated human calcineurin Aα, respectively.

[0081]FIG. 4A is a sequence alignment comparing the calcineurin-dockingsequences present in the NFAT family of proteins. Consensus=consensussequence of the four NFAT sequences shown.

[0082]FIG. 4B depicts an amino acid sequence and a first round ofselection for an optimized peptide inhibitor. Particular amino acidsequences selected in the degenerate positions are shown with preferencevalues indicated in parenthesis. Residues showing strong selection areunderlined.

[0083]FIG. 4C depicts an amino acid sequence and a second round ofselection for an optimized peptide inhibitor. An alternative set ofresidues was chosen based on the initial screen shown in FIG. 4B toorient a secondary library, and the second library was selected onGST-calcineurin (amino acids 2 to 347) to derive high affinity peptides.Residues locked in from the screening in FIG. B are boxed. “Z” indicatesa set of non-natural amino acids.

[0084]FIG. 4D depicts the amino acid sequence of the VIVIT peptide.

[0085]FIG. 5A is a Western blot that illustrates that SPRIEIT and VIVITpeptide inhibit the NFAT-calcineurin interaction. Cn=calcineurin,CaM=calmodulin, Ca2+=CaCl₂, Cn A=calcineurin A chain.

[0086]FIG. 5B is a Western blot that illustrates that that SPRIEIT andVIVIT peptide inhibit calcineurin-mediated dephosphorylation of NFAT.The positions of phospho-and dephosphoNFAT1 are indicated by arrows.Cn=calcineurin, CaM=calmodulin, Ca2+=CaCl₂, NaPPi=sodium pyrophosphate.

[0087]FIG. 5C is a bar graph illustrating that SPRIEIT and VIVITpeptides do not block dephosphorylation of RII phosphopeptide bycalcineurin. The numbers next to each peptide label indicate peptideconcentrations in μM. CsA/CypA complexes were used at 10 μM.CsA/Cyp=cyclosporin A/cyclophilin A.

[0088]FIG. 6A is a bar graph illustrating that VIVIT and SPRIEITpeptide, delivered intracellularly as a GFP-VIVIT and GFP-SPRIEIT fusionproteins, respectively, block expression of an NFAT-dependent reportergene. Endog.=endogenous NFAT; NFAT1=overexpressed human NFAT1;NFAT2=overexpressed human NFAT2; NFAT4=overexpressed human NFAT4;PMA+iono=PMA and ionomycin stimulated cells; unstimulated=cells notstimulated with PMA and ionomycin.

[0089]FIG. 6B is a set of bar graphs illustrating that VIVIT peptide,delivered intracellularly as a GFP-VIVIT fusion protein, blocksexpression of an NFAT-dependent reporter gene, but not of anNF-κB-dependent reporter gene. 3×NFAT-Luc=reporter plasmid containingthree tandem NFAT sites in a promoter controlling a luciferase gene;2×NF-κB-Luc=reporter plasmid containing two NF-κB sites in a promotercontrolling a luciferase gene; PMA+iono=PMA and ionomycin stimulatedcells; Unstim.=cells not stimulated with PMA and ionomycin.

[0090]FIG. 6C is a set of bar graphs illustrating that cyclosporin Ablocks expression of both reporter genes used in FIG. 6B.3×NFAT-Luc=reporter plasmid containing three tandem NFAT sites in apromoter controlling a luciferase gene; 2×NF-κB-Luc=reporter plasmidcontaining two NF-κB sites in a promoter controlling a luciferase gene;CsA=cyclosporin A; P+I=PMA plus ionomycin.

[0091]FIG. 6D is a set of bar graphs that illustrate that the VIVITpeptide, delivered intracellularly as a GFP-VIVIT fusion protein,inhibits reporter activity driven by IL-2 and TNF-α promoters. IL-2promoter=reporter plasmid containing human IL-2 promoter controlling aluciferase gene; TNF-α promoter=reporter plasmid containing human TNF-αpromoter controlling a luciferase gene; PMA+iono=PMA and ionomycinstimulated cells; Unstim.=cells neither stimulated with PMA andionomycin nor stimulated with antibodies; CD3+CD28=cells stimulated withanti-CD3 and anti-CD28 antibodies.

[0092]FIG. 7A is a picture of an RNA gel autoradiogram illustrating thatT cells expressing GFP-VIVIT displayed attenuated induction of IL-2 andIL-13 mRNAs.

[0093]FIG. 7B is a picture of an RNA gel autoradiogram illustrating thatT cells expressing GFP-VIVIT display attenuated induction of IL-3, TNFα,GM-CSF, and MIP-1α mRNAs.

[0094]FIG. 7C is a picture of an RNA gel autoradiogram illustrating thatT cells expressing GFP-VIVIT display attenuated induction TNFα.

[0095]FIG. 8A is a line graph illustrating saturable binding of afluorescent VIVIT peptide to calcineurin.

[0096]FIG. 8B is a line graph illustrating competition for binding tocalcineurin by unlabelled VIVIT peptide.

[0097]FIG. 9A is a line graph illustrating that the organic compoundsINCA1 and INCA2 compete with a VIVIT peptide for binding to calcineurin.

[0098]FIG. 9B is a line graph illustrating that the organic compoundINCA6 competes with a VIVIT peptide for binding to calcineurin.

[0099]FIG. 10A is an NMR spectrum illustrating that INCA1 binds tocalcineurin.

[0100]FIG. 10B is an NMR spectrum illustrating that INCA2 binds tocalcineurin.

[0101]FIG. 10C is an NMR spectrum illustrating that INCA6 binds tocalcineurin.

[0102]FIG. 11A is a picture of a Western Blot illustrating that INCA2blocks dephosphorylation of NFAT by calcineurin in vitro.Iono=ionomycin. PPi=sodium pyrophosphate; CN=calcineurin in the absenceof inhibitor; DMSO=Dimethyl Sulfoxide.

[0103]FIG. 11B is a bar graph illustrating that INCA compounds do notblock dephosphorylation of RII phosphopeptide. DMSO=Dimethyl Sulfoxide;CsA/Cyp=cyclosporin A/cyclophilin A.

[0104]FIG. 12A is a picture of a Western blot illustrating that INCA6blocks dephosphorylation of NFAT by calcineurin in cells.Iono=ionomycin.

[0105]FIG. 12B is a picture of a Western blot illustrating that INCA6does not block phosphorylation of MAP kinase. PMA=phorbol 12-myristate13-acetate.

[0106]FIG. 13 is a set of micrographs illustrating that INCA6 blocks thenuclear import of NFAT in cells. Iono=ionomycin; CsA=cyclosporin A.

[0107]FIG. 14A is a picture of an RNA gel autoradiogram illustratingthat INCA6 attenuated induction of GM-CSF in T cells.CsA/FK506=cyclosporin A and FK506; Iono=ionomycin; PMA=phorbol12-myristate 13-acetate.

[0108]FIG. 14B is a picture of an RNA gel autoradiogram illustratingthat INCA6 attenuated induction of TNFα and IFNγ in T cells.CsA/FK506=cyclosporin A and FK506; Iono=ionomycin; PMA=phorbol12-myristate 13-acetate.

[0109]FIG. 14C is a picture of an RNA gel autoradiogram illustratingthat INCA6 attenuated induction of lymphotactin (Ltn), MIP-1β, andMIP-1α in T cells. CsA/FK506=cyclosporin A and FK506; Iono=ionomycin;PMA=phorbol 12-myristate 13-acetate.

[0110]FIG. 15A illustrates the general chemical structure of INCA1,typical chemical modifications that can be made thereto, and theinhibitory activity exhibited by the modified compounds.

[0111]FIG. 15B illustrates the general chemical structure of INCA2,typical chemical modifications that can be made thereto, and theinhibitory activity exhibited by the modified compounds.

[0112]FIG. 15C illustrates the general chemical structure of INCA6,typical chemical modifications that can be made thereto, and theinhibitory activity exhibited by the modified compounds.

DETAILED DESCRIPTION

[0113] This invention provides an isolated fragment of the conservedregulatory domain of NFAT protein capable of inhibiting protein-proteininteraction between calcineurin and NFAT, or a biologically activeanalog thereof.

[0114] By NFAT protein (nuclear factor of activated T cells) is meant amember of a family of transcription factors comprising the membersNFAT1, NFAT2, NFAT3 and NFAT4, with several isoforms. Any other NFATprotein whose activation is calcineurin dependent is also meant to beincluded. NFAT proteins can be, e.g., mammalian proteins, e.g., human ormurine. NFAT1, NFAT2 and NFAT4 are expressed in immune cells, e.g., Tlymphocytes, and play a role in eliciting immune responses. NFATproteins are involved in the transcriptional regulation of cytokinegenes, e.g., IL-2, IL-3, IL-4, TNF-α and IFN-γ, during the immuneresponse.

[0115] cDNA sequences for NFAT have been previously reported. SeeMcCaffrey et al., Science 262:750-754 (1993) and Luo et al., Mol. CellBiol. 16:3955-3966 (1996) for murine NFAT1. See Luo et al., Mol. CellBiol. 16:3955-3966 (1996) for human NFAT1. See Northrop et al., Nature369:497-502 (1994) for human NFAT2, and Park et al., J. Biol. Chem.271:20914-20921 (1996) for human NFAT2b. The published sequences forhuman NFAT2 represent two isoforms differing by alternative splicing atthe N and C termini, but having the same regulatory domain andDNA-binding domain. See Hoey et al., Immunity 2:461-472 (1995) for humanNFAT3. See Masuda et al., Mol. Cell Biol. 15:2697-2706 (1995) and Hoeyet al., Immunity 2:461-472 (1995) for human NFAT4. See Ho et al., J.Biol. Chem. 270:19898-19907 (1995) and Liu et al., Mol. Cell Biol.8:157-170 (1997) for murine NFAT4. The two published sequences formurine NFAT4 are not identical.

[0116] NFAT proteins have been shown to be direct substrates ofcalcineurin. Calcineurin is a calmodulin-dependent, cyclosporinA/FK506-sensitive, phosphatase. Calcineurin is activated through itsinteraction with Ca⁺² activated calmodulin when intracellular calciumlevels are elevated as a result of receptor crosslinking andphospholipase C activation. The activated calcineurin in turn activatesNFAT from an inactive cytoplasmic pool. NFAT activation involvesprotein-protein interaction between calcineurin and NFAT,dephosphorylation of NFAT by calcineurin, conformational change in NFATresulting from the interaction between calcineurin and NFAT or thedephosphorylation of NFAT and translocation of NFAT to the nucleus. NFATactivation results in induction of NFAT-dependent gene expression of,e.g., cytokine genes.

[0117] The conserved regulatory domain of NFAT is an N-terminal regionof NFAT which is about 300 amino acids in length. The conservedregulatory domain of murine NFAT1 is a region extending from amino acidresidue 100 through amino acid residue 397, of human NFAT1 is a regionextending from amino acid residue 100 through 395, of human NFAT2 is aregion extending from amino acid residue 106 through 413, of humanNFAT2b is a region extending from amino acid residue 93 through 400, ofhuman NFAT3 is a region extending from amino acid residue 102 through404, and of human NFAT4 is a region extending from amino acid residue 97through 418. The conserved regulatory domain is moderately conservedamong the members of the NFAT family, NFAT1, NFAT2, NFAT3 and NFAT4. Theconserved regulatory region binds directly to calcineurin. The conservedregulatory region is located immediately N-terminal to the DNA-bindingdomain (amino acid residues 398 through 680 in murine NFAT1, amino acidresidues 396 through 678 in human NFAT1, amino acid residues 414 through696 in human NFAT2, amino acid residues 401 through 683 in human NFAT2b,amino acid residues 405 through 686 in human NFAT3, and amino acidresidues 419 through 700 in human NFAT4).

[0118] In certain embodiments of the invention, the peptide fragment orbiologically active analog thereof is further capable of inhibitingdephosphorylation of NFAT by calcineurin. In certain embodiments, thepeptide fragment or biologically active analog thereof is furthercapable of inhibiting recruitment of NFAT to the nucleus in a cell. Incertain embodiments, the peptide fragment or biologically active analogthereof is further capable of inhibiting conformational change in NFATthat results from the protein-protein interaction between NFAT andcalcineurin or from the dephosphorylation of NFAT by calcineurin. Incertain embodiments, the peptide fragment or biologically active analogthereof is further capable of inhibiting NFAT-dependent genetranscription.

[0119] Preferably, the peptide fragment or biologically active analogthereof does not inhibit or does not substantially inhibit the activityof calcineurin toward non-NFAT calcineurin substrates. Calcineurinnormally is capable of interacting with many different substrates, e.g.,NFAT and the microtubule-associated protein tau (Fleming and Johnson,Biochem J 309:41-47 (1995); Yamamoto et al, J Biochem 118:1224-1231(1995)), the regulatory subunit RII of cAMP-dependent protein kinase(Blumenthal and Krebs, Biophys J 41:409a (1983)), inhibitor-1 (Hemmingset al, Nature 310:503-505 (1984); Mulkey et al, Nature 369:486-488(1994)), dopamine-and cAMP-regulated phosphoprotein DARPP-32 (Hemmingset al, Nature 310:503-505 (1984)), a dihydropyridine-sensitivevoltage-dependent Ca²⁺ channel (Hosey et al, Proc Natl Acad Sci USA83:3733-3737 (1986)), nitric oxide synthase (Dawson et al, Proc NatlAcad Sci USA 90:9808-9812 (1993)), dynamin (Liu et al, Science265:970-973 (1994); Nichols et al, J Biol Chem 269:23817-23823 (1994)),the inositol 1, 4, 5-trisphosphate receptor-FKBP12 complex (Cameron etal, Cell 83:463-472 (1995)), and the ryanodine receptor-FKBP12 complex(Cameron et al, Cell 83:463-472 (1995)). A key advantage of thisinvention is that it includes peptide fragments and analogs thereofwhich are specific for the interaction between calcineurin and NFAT.Such specific inhibitors can be used for therapeutic purposes withreduced toxic effects, as compared to general immunosuppressants.

[0120] By fragment of the conserved regulatory domain of NFAT protein ismeant some portion of the naturally occurring conserved regulatorydomain of NFAT protein. Preferably, the fragment is less than about 150amino acid residues, more preferably is less than about 100 amino acidresidues, more preferably yet is less than about 50 amino acid residues,more preferably yet is less than about 30 amino acid residues, morepreferably yet is less than about 20 amino acid residues, morepreferably yet is less than about 10 amino acid residues, and mostpreferably is less than about 6 amino acid residues in length.Preferably, the fragment is greater than about 3 amino acid residues inlength. Fragments include, e.g., truncated secreted forms, cleavedfragments, proteolytic fragments, splicing fragments, other fragments,and chimeric constructs between at least a portion of the relevant geneand another molecule. Fragments of the conserved regulatory domain ofNFAT protein can be generated by methods known to those skilled in theart. In preferred embodiments, the fragment is biologically active. Theability of a candidate fragment to exhibit a biological activity of theconserved regulatory domain of NFAT can be assessed by, e.g., itsability to form a protein-protein interaction with calcineurin, or itsability to inhibit the binding of NFAT to calcineurin, by methods asdescribed herein. Also included are fragments containing residues thatare not required for biological activity of the fragment or that resultfrom alternative mRNA splicing or alternative protein processing events.

[0121] Fragments of a protein can be produced by any of a variety ofmethods known to those skilled in the art, e.g., recombinantly, byproteolytic digestion, or by chemical synthesis. Internal or terminalfragments of a polypeptide can be generated by removing one or morenucleotides from one end (for a terminal fragment) or both ends (for aninternal fragment) of a nucleic acid which encodes the polypeptide.Expression of the mutagenized DNA produces polypeptide fragments.Digestion with “end-nibbling” endonucleases can thus generate DNAs whichencode an array of fragments. DNAs which encode fragments of a proteincan also be generated, e.g., by random shearing, restriction digestion,chemical synthesis of oligonucleotides, amplification of DNA using thepolymerase chain reaction, or a combination of the above-discussedmethods.

[0122] Fragments can also be chemically synthesized using techniquesknown in the art, e.g., conventional Merrifield solid phase f-Moc ort-Boc chemistry. For example, peptides of the present invention can bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

[0123] An NFAT protein used for generating analogs or fragments can beobtained, e.g., from purification or secretion of a naturally occurringNFAT protein, from recombinant NFAT protein, or from synthesized NFATprotein.

[0124] In certain embodiments, the peptide fragment comprises the aminoacid sequence IX₂X₃T (SEQ ID NO:104), wherein X₂ is E, R or Q, and X₃ isI or F. Preferred amino acid sequences are, e.g., IEIT (SEQ ID NO:105),IRIT (SEQ ID NO:106), IQIT (SEQ ID NO:107), and IQFT (SEQ ID NO:108).

[0125] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₁IX₂X₃T (SEQ ID NO:73), wherein X₁ is R or S, X₂ is E, Ror Q, and X₃ is I or F. Preferred amino acid sequences are, e.g.,X₁IX₂IT (SEQ ID NO:74), RIX₂IT (SEQ ID NO:75), X₁IEIT (SEQ ID NO:76),RIEIT (SEQ ID NO:1), SIRIT (SEQ ID NO:2), SIQIT (SEQ ID NO:3), and SIQFT(SEQ. ID NO:4).

[0126] In certain embodiments, the peptide fragment comprises the aminoacid sequence PX₁IX₂X₃T (SEQ ID NO:77), wherein X₁ is R or S, X₂ is E, Ror Q, and X₃ is I or F. Preferred amino acid sequences are, e.g., PRIEIT(SEQ ID NO:5), PSIRIT (SEQ ID NO:6), PSIQIT (SEQ ID NO:71) and PSIQFT(SEQ ID NO:7).

[0127] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₅PX₁IX₂X₃T (SEQ ID NO:78), wherein X₁ is R or S, X₂ is E,R or Q, X₃ is I or F and X₅ is S or C. Preferred amino acid sequencesare, e.g., SPRIEIT (SEQ ID NO:8), CPSIRIT (SEQ ID NO:9), CPSIQIT (SEQ IDNO:10) and CPSIQFT (SEQ ID NO:11).

[0128] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₅PX₁IX₂X₃TX₆ (SEQ ID NO:79), wherein X₁ is R or S, X₂ isE, R or Q, X₃ is I or F, X₅ is S or C, and X₆ is P or S. Preferred aminoacid sequences are, e.g., SPRIEITP (SEQ ID NO:12), SPRIEITS (SEQ IDNO:13), CPSIRITS (SEQ ID NO:14), CPSIQITS (SEQ ID NO:15) and CPSIQFTS(SEQ ID NO:16).

[0129] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₅PX₁IX₂X₃TX₆X₇ (SEQ ID NO:80), wherein X₁ is R or S, X₂is E, R or Q, X₃ is I or F, X₅ is S or C, X₆ is P or S, and X₇ is S, Cor I. Preferred amino acid sequences are SPRIEITPS, (SEQ ID NO:17)SPRIEITSC, (SEQ ID NO:18) CPSTRITSI, SEQ ID NO:19) CPSIQITSI and (SEQ IDNO:20) CPSIQFTSI. (SEQ ID NO:21)

[0130] In certain embodiments, the peptide fragment comprises the aminoacid sequence X₁₁IX₁₀X₉X₅PX₁IX₂X₃TX₆X₇X₈ (SEQ ID NO:81), wherein X₁ is Ror S, X₂ is E, R or Q, X₃ is I or F, X₅ is S or C, X₆ is P or S, X₇ isS, C or I, X₈ is H, L or S, X₉ is P, L or E, X₁₀, is G, L or F, and X₁₁is S, A, V or P. Preferred amino acid sequences are, e.g., SGPSPRIEITPSH(SEQ ID NO:22), SGLSPRIEITPSH (SEQ ID NO:23), ALESPRIEITSCL (SEQ IDNO:24), VLECPSIRITSIS (SEQ ID NO:25), PFECPSIQITSIS (SEQ ID NO:26),PFECPSIQITSIS (SEQ ID NO:27) and PFECPSIQFTSIS (SEQ ID NO:28). Otherpreferred amino acid sequences are, e.g., KPAGASGPSPRIEITPSHELMQAGG,(SEQ ID NO:29) KPAGASGLSPRIEITPSHELIQAVG, (SEQ ID NO:30)PDGAPALESPRIEITSCLGLYHNNN, (SEQ ID NO:31) AGGGRVLECPSIRITSISPTPEPPA,(SEQ ID NO:32) LGGPKPFECPSIQITSISPNCHQEL, (SEQ ID NO:33)LGGPKPFECPSIQITSISPNCHQGT and (SEQ ID NO:34) LGGPKPFECPSIQFTSISPNCQQEL.(SEQ ID NO:35)

[0131] By a biologically active analog of the NFAT fragment is meant ananalog that is capable of inhibiting protein-protein interaction betweencalcineurin and NFAT.

[0132] By analog is meant a compound that differs from the naturallyoccurring NFAT fragment in amino acid sequence or in ways that do notinvolve sequence, or both. Peptide analogs of the invention generallyexhibit at least about 70% homology, preferably at least about 80%homology, more preferably at least about 90% homology, more preferablyyet at least about 95% homology, more preferably yet at least about 97%homology, and most preferably at least about 98% homology, withsubstantially the entire sequence of a naturally occurring NFATfragment, preferably with a segment of about 150 amino acid residues,more preferably with a segment of about 100 amino acid residues, morepreferably yet with a segment of about 50 amino acid residues, morepreferably yet with a segment of about 30 amino acid residues, morepreferably yet with a segment of about 20 amino acid residues, morepreferably yet with a segment of about 10 amino acid residues, morepreferably yet with a segment of about 5 amino acid residues, and mostpreferably yet with a segment of about 4 amino acid residues.Non-sequence modifications include, e.g., in vivo or in vitro chemicalderivatizations of the NFAT fragment. Non-sequence modificationsinclude, e.g., changes in phosphorylation, acetylation, methylation,carboxylation, or glycosylation. Methods for making such modificationsare known to those skilled in the art. For example, phosphorylation canbe modified by exposing the peptide to phosphorylation-altering enzymes,e.g., kinases or phosphatases.

[0133] Preferred analogs include an NFAT fragment whose sequence differsfrom the wild-type sequence by one or more conservative amino acidsubstitutions or by one or more non-conservative amino acidsubstitutions, deletions, or insertions, which do not abolish biologicalactivity of the peptide. Conservative substitutions typically includethe substitution of one amino acid for another with similarcharacteristics, e.g., substitutions within the following groups:valine, glycine; glycine, alanine; valine, isoleucine, leucine; asparticacid, glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. Other examples of conservativesubstitutions are shown in Table 1. TABLE 1 CONSERVATIVE AMINO ACIDSUBSTITUTIONS For Amino Acid Code Replace with any of Alanine A D-Ala,Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg,D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp,D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic D D-Asp, D-Asn, Asn, Glu, D-Glu,Gln, D-Gln Acid Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-ThrGlutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic E D-Glu,D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Acid Glycine G Ala, D-Ala, Pro,D-Pro, P-Ala Acp Histidine H D-His Isoleucine I D-Ile, Val, D-Val, Leu,D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-MetLysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile,D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu,Val, D-Val Phenyl- F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His; Trp, alanineD-Trp, Trans-3, 4, or 5-phenylproline, cis-3, 4, or 5-phenylprolineProline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-orL-I-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr,Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser,D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tryptophan WD-Trp, Phe, D-Phe, Tyr, D-Tyr Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0134] Amino acid sequence variants of a protein can be prepared by anyof a variety of methods known to those skilled in the art. For example,random mutagenesis of DNA which encodes a protein or a particular domainor region of a protein can be used, e.g., PCR mutagenesis (using, e.g.,reduced Taq polymerase fidelity to introduce random mutations into acloned fragment of DNA; Leung et al., BioTechnique 1:11-15 (1989)), orsaturation mutagenesis (by, e.g., chemical treatment or irradiation ofsingle-stranded DNA in vitro, and synthesis of a complementary DNAstrand; Mayers et al., Science 229:242 (1985)). Random mutagenesis canalso be accomplished by, e.g., degenerate oligonucleotide generation(using, e.g., an automatic DNA synthesizer to chemically synthesizedegenerate sequences; Narang, Tetrahedron 39:3 (1983); Itakura et al.,Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A. G.Walton, Amsterdam: Elsevier, pp. 273-289 (1981)). Non-random or directedmutagenesis can be used to provide specific sequences or mutations inspecific regions. These techniques can be used to create variants whichinclude, e.g., deletions, insertions, or substitutions, of residues ofthe known amino acid sequence of a protein. The sites for mutation canbe modified individually or in series, e.g., by (i) substituting firstwith conserved amino acids and then with more radical choices dependingupon results achieved, (ii) deleting the target residue, (iii) insertingresidues of the same or a different class adjacent to the located site,or (iv) combinations of the above.

[0135] Methods for identifying desirable mutations include, e.g.,alanine scanning mutagenesis (Cunningham and Wells, Science244:1081-1085 (1989)), oligonucleotide-mediated mutagenesis (Adelman etal., DNA 2:183 (1983)), cassette mutagenesis (Wells et al., Gene 34:315(1985)), combinatorial mutagenesis, and phage display libraries (Ladneret al., PCT International Appln. No. WO88/06630). The NFAT fragmentanalogs can be tested in physical and/or functional assays, e.g., intheir ability to inhibit protein-protein interaction between calcineurinand NFAT, as described herein.

[0136] Other analogs within the invention include, e.g., those withmodifications which increase peptide stability. Such analogs cancontain, e.g., one or more non-peptide bonds (which replace the peptidebonds) in the peptide sequence. Also included are, e.g., analogs thatinclude residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,β or γ amino acids and cyclic analogs.

[0137] Analogs are also meant to include peptides in which structuralmodifications have been introduced into the peptide backbone so as tomake the peptide non-hydrolyzable. Such peptides are particularly usefulfor oral administration, as they are not digested. Peptide backbonemodifications include, e.g., modifications of the amide nitrogen, theα-carbon, the amide carbonyl, or the amide bond, and modificationsinvolving extensions, deletions or backbone crosslinks. For example, thebackbone can be modified by substitution of a sulfoxide for thecarbonyl, by reversing the peptide bond, or by substituting a methylenefor the carbonyl group. Such modifications can be made by standardprocedures known to those skilled in the art. See, e.g., Spatola, A. F.,“Peptide Backbone Modifications: A Structure-Activity Analysis ofPeptides Containing Amide Bond Surrogates, Conformational Constraints,and Related Backbone Replacements,” in Chemistry and Biochemistry ofAmino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein(ed.), Marcel Dekker, Inc., New York (1983).

[0138] An analog is also meant to include polypeptides in which one ormore of the amino acid residues include a substituent group, orpolypeptides which are fused with another compound, e.g., a compound toincrease the half-life of the polypeptide, e.g., polyethylene glycol.

[0139] Analogs are also meant to include those produced by introductionof amino acid substitutions, or the design of constrained peptides,cyclic peptides, and other modified peptides or analogs, where themodifications or constraints are introduced on the basis of knowledge ofthe conformation of the peptide bound to calcineurin, or on the basis ofknowledge of the structure of a protein-protein complex formed by NFATand calcineurin or by a fragment of NFAT (including, e.g., the 13-merpeptide described herein) and a fragment of calcineurin. Theconformation of the bound peptide can be determined by techniques knownto those skilled in the art, e.g., NMR, e.g., transferred nuclearOverhauser effect spectroscopy (transferred NOESY) of a rapidlydissociating peptide to determine distance constraints (Campbell andSykes, J. Magn. Reson. 93:77-92 (1991); Lian et al., Methods Enzymol.239:657-700 (1994)), with or without additional NMR techniques, followedby the use of the distance constraints and of constrained moleculardynamics simulations and energy minimization with available computersoftware (e.g., the NMR_Refine module of the InsightII™ suite ofprograms (Biosym/MSI, San Diego, Calif.), and the Discover™ or Discover3.0™ molecular simulation programs (Biosym/MSI, San Diego, Calif.)) toarrive at a structural model. Alternatively, the structure of thespecified complexes, including the conformation assumed by the claimedpeptides in the complex, can be determined by x-ray crystallography.

[0140] Other analogs within the scope of the invention include compoundsin which the peptide fragment or biologically active analog thereof iscovalently linked to a ligand that binds to a site adjacent to thatrecognized by the 13-mer peptide described herein (See Shuker et al.,Science 274:1531-1534 (1996)), in order to produce a biologically activecompound with increased affinity or specificity for the calcineurin-NFATinteraction.

[0141] The invention also includes an isolated polynucleotide encodingthe peptide comprising the amino acid sequence as set forth in SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, or biologically activeanalogs thereof. The invention also includes an isolated polynucleotideencoding the peptide comprising the amino acid sequence as set forth inSEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, orbiologically active analogs thereof. Preferred polynucleotide sequencesare, e.g., the sequences as set forth in SEQ ID NO:109, SEQ ID NO:110,SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 and SEQ IDNO:115.

[0142] The invention also includes an isolated polynucleotide encodingthe peptide comprising the amino acid sequence as set forth in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or biologically activeanalogs thereof. Preferred polynucleotide sequences are, e.g., thesequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQID NO:39, SEQ ID NO:83 and SEQ ID NO:84.

[0143] The invention also includes an isolated polynucleotide encodingthe peptide comprising the amino acid sequence as set forth in SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, orbiologically active analogs thereof.

[0144] The invention also includes an isolated polynucleotide encodingthe peptide comprising the amino acid sequence as set forth in SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:71, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, or biologically active analogs thereof.Preferred polynucleotide sequences are, e.g., the sequences as set forthin SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42, SEQ ID NO:43,SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85, SEQ ID NO:86,SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 andSEQ ID NO:92.

[0145] The invention also includes an isolated polynucleotide encodingthe peptide comprising the amino acid sequence as set forth in SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, or biologically active analogs thereof. Preferredpolynucleotide sequences are, e.g., the sequences as set forth in SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62 and SEQ ID NO:63.

[0146] The invention also includes an isolated polynucleotide encodingthe peptide comprising the amino acid sequence as set forth in SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, or biologically active analogs thereof. Preferredpolynucleotide sequences are, e.g., sequences as set forth in SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69 and SEQ ID NO:70.

[0147] The invention also includes nucleotide sequences which arecapable of hybridizing to and which are at least about 70%, preferablyat least about 80%, more preferably yet at least about 85%, morepreferably yet at least about 90%, more preferably yet at least about95%, more preferably yet at least about 97%, and most preferably atleast about 98% identical to the polynucleotides described herein, andwhich encode a peptide having biological activity. By percent identityis meant the maximal percent identity obtained by aligning the firstbase of the oligonucleotide with any base in the nucleotide sequence andthen scoring the identity of aligned bases for each base in theoligonucleotide without introduction of any gaps.

[0148] The nucleotide sequences of the present invention can be in theform of, e.g., RNA, DNA or PNA, e.g., cRNA, cDNA, genomic DNA, or e.g.,synthetic RNA, DNA or PNA. The nucleotide sequence can bedouble-stranded or single stranded, and if single stranded can be thecoding strand or non-coding (anti-sense) strand.

[0149] The coding sequence which encodes the peptide fragments can beidentical to the coding sequences as set forth in SEQ ID NOS:36-70, 72,83-92 or 110-115, or can be a different coding sequence, which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same peptide fragments as the nucleic acid as setforth in SEQ ID NOS:36-70, 72, 83-92 or 110-115.

[0150] The invention also includes a gene therapy vector comprising anucleotide sequence encoding a peptide fragment of the conservedregulatory domain of NFAT protein capable of inhibiting protein-proteininteraction between calcineurin and NFAT, or a biologically activeanalog of the peptide fragment.

[0151] By a gene therapy vector is meant a vector useful for genetherapy. Gene therapy vectors carry a gene of interest that is usefulfor gene therapy. The gene therapy vectors are able to be transferred tothe cells of an animal, e.g., a human, and are able to express the geneof interest in such cells so as to effect gene therapy. The vector canbe, e.g., chromosomal, nonchromosomal or synthetic. It can be, e.g., RNAor DNA. The vector can be, e.g., a plasmid, a virus or a phage.Preferred vectors include, e.g., retroviral vectors, adenoviral vectors,adeno-associated vectors, herpes virus vectors and Semliki Forest virusvector. A preferred retroviral vector is Murine Stem Cell Virus (MSCV),which is a variant of Moloney Murine Leukemia Virus (MoMLV).

[0152] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107,SEQ ID NO:108, or biologically active analogs thereof. In certainembodiments, the gene therapy vector comprises the nucleotide sequencesas set forth in SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ IDNO:113, SEQ ID NO:114 or SEQ ID NO:115. The invention also includesnucleotide sequences which are capable of hybridizing to and which areat least about 70%, preferably at least about 80%, more preferably yetat least about 85%, more preferably yet at least about 90%, morepreferably yet at least about 95%, more preferably yet at least about97%, and most preferably at least about 98% identical to thesenucleotide sequences, and which encode a peptide having biologicalactivity.

[0153] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, or biologically active analogs thereof. In certain embodiments,the gene therapy vector comprises the nucleotide sequences as set forthin SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:83or SEQ ID NO:84. The invention also includes nucleotide sequences whichare capable of hybridizing to and which are at least about 70%,preferably at least about 80%, more preferably yet at least about 85%,more preferably yet at least about 90%, more preferably yet at leastabout 95%, more preferably yet at least about 97%, and most preferablyat least about 98% identical to these nucleotide sequences, and whichencode a peptide having biological activity. In preferred embodiments,the gene therapy vector comprises a nucleotide sequence encoding thepeptide comprising the amino acid sequence as set forth in SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:71, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, or biologically active analogs thereof. In certainembodiments, the gene therapy vector comprises the nucleotide sequenceas set forth in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:85,SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90,SEQ ID NO:91 or SEQ ID NO:92. The invention also includes nucleotidesequences which are capable of hybridizing to and which are at leastabout 70%, preferably at least about 80%, more preferably yet at leastabout 85%, more preferably yet at least about 90%, more preferably yetat least about 95%, more preferably yet at least about 97%, and mostpreferably at least about 98% identical to these nucleotide sequences,and which encode a peptide having biological activity. In preferredembodiments, the gene therapy vector comprises a nucleotide sequenceencoding the peptide comprising the amino acid sequence as set forth inSEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, or biologically active analogs thereof. Incertain embodiments, the gene therapy vector comprises the nucleotidesequence as set forth in SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63. The invention alsoincludes nucleotide sequences which are capable of hybridizing to andwhich are at least about 70%, preferably at least about 80%, morepreferably yet at least about 85%, more preferably yet at least about90%, more preferably yet at least about 95%, more preferably yet atleast about 97%, and most preferably at least about 98% identical tothese nucleotide sequences, and which encode a peptide having biologicalactivity.

[0154] In preferred embodiments, the gene therapy vector comprises anucleotide sequence encoding the peptide comprising the amino acidsequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID SEQ ID NO:34 and SEQ ID NO:35, orbiologically active analogs thereof. In certain embodiments, the genetherapy vector comprises the nucleotide sequence as set forth in SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69 or SEQ ID NO:70. The invention also includes nucleotide sequenceswhich are capable of hybridizing to and which are at least about 70%,preferably at least about 80%, more preferably yet at least about 85%,more preferably yet at least about 90%, more preferably yet at leastabout 95%, more preferably yet at least about 97%, and most preferablyat least about 98% identical to these nucleotide sequences, and whichencode a peptide having biological activity.

[0155] In certain embodiments, the gene therapy vector also has anucleotide sequence encoding a signal peptide for effecting secretion ofthe NFAT peptide fragment. Examples of signal peptides include those of(pre)prolactin (Walter and Blobel, J Cell Biol 91:557-561(1981)),apolipoprotein AI (Stoffel et al, Eur J Biochem 120:519-522 (1981)), andβ-lactamase (Muller et al, J Biol Chem 257:11860-11863 (1982)).Preferably, the signal peptide is encoded 5′ of the nucleotide sequenceencoding the NFAT peptide fragment.

[0156] In certain embodiments, the gene therapy vector has a nucleotidesequence encoding a tag for identification of the NFAT peptide fragment.Examples of tags that can be detected with commercially availableantibodies (Shiio et al, Methods Enzymol 254:497-502 (1995)) are theFLAG peptide, the peptide YPYDVPDYA (SEQ ID NO:93) from influenza virushaemagglutinin, and other peptides from T7 gene 10 protein, Myc, andbovine papillomavirus L1 protein. The sequence encoding the tag can be5′ or 3′ of the nucleotide sequence encoding the NFAT peptide fragment.

[0157] In certain embodiments the gene therapy vector has a selectablemarker. Examples of selectable markers include a Neomycinphosphotransferase gene, a humanized red-shifted green fluorescentprotein, hygromycin resistance, puromycin resistance, luciferase, or acell-surface protein that is recognized by a specific monoclonalantibody.

[0158] In certain embodiments, the gene therapy vector has an induciblepromoter, e.g., a promoter that will allow expression of the therapeuticpeptide at a specific time or in a graded manner. Such a construct isvaluable, e.g., for the purpose of treating graft-versus-host diseaseafter a transplant of bone marrow cells or stem cells geneticallyengineered to carry the gene therapy vector with a promoter inducible bya compound that can be administered orally; or for cell therapy ofmultiple sclerosis with glial cells genetically engineered to express animmunosuppressive protein or peptide in response to a cytokine or othermolecule produced at a site of autoimmune demyelination.

[0159] In certain embodiments, the gene therapy vector has acell-specific promoter to allow inhibition of the calcineurin-NFATinteraction in one cell type, without disturbing normal NFAT function inother types of cells.

[0160] The invention also includes a cell having a gene therapy vectordescribed herein. Preferably, the cell is an animal cell. The genetherapy vectors described herein can be introduced into a cell, e.g., bytransformation, transfection, transduction, infection, or ex vivoinjection. Preferably, they are targeted to a particular cell type orcell.

[0161] The invention also includes a method for producing a peptidecapable of inhibiting protein-protein interaction between calcineurinand NFAT, comprising culturing a cell having a gene therapy vectordescribed herein under conditions that permit expression of the peptide.

[0162] The invention also includes a method for treating an immunerelated disease or condition in an animal. Immune-related diseases orconditions include, e.g., acute immune diseases, chronic immune diseasesand autoimmune diseases. It is also meant to include treatment of tissueor organ transplant graft rejections or graft-versus-host disease. Agene therapy vector described herein is administered to the animal.

[0163] The invention also includes a method for providing an animalhaving an immune-related disease or condition with a therapeuticallyeffective level of a peptide capable of inhibiting protein-proteininteraction between calcineurin and NFAT. A gene therapy vectordescribed herein is administered to the animal.

[0164] The invention also includes a method for inhibiting an immuneresponse in an animal. An animal in need of inhibition of an immuneresponse is provided. A therapeutically effective amount of a peptidefragment of the conserved regulatory domain of NFAT protein capable ofinhibiting protein-protein interaction between calcineurin and NFAT, ora biologically active analog thereof, is provided. The peptide fragmentor biologically active analog thereof is administered to the animal soas to inhibit the immune response in the animal.

[0165] The peptide fragment can be any of the peptide fragments of theconserved regulatory domain of NFAT protein of this invention describedherein. Certain preferred peptide fragments comprise the amino acidsequence X₁IX₂X₃T (SEQ ID NO:73) or a biologically active analogthereof, wherein X₁ is R or S, X₂ is E, R or Q, and X₃ is I or F. Incertain embodiments, the peptide fragment comprises the amino acidsequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, or biologically active analogs thereof.

[0166] Other preferred peptide fragments comprise the amino acidsequence IX₂X₃T (SEQ ID NO:104) or a biologically active analog thereof,wherein X₂ is E, R or Q, and X₃ is I or F. In certain embodiments, thepeptide fragment comprises the amino acid sequence as set forth in SEQID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologicallyactive analogs thereof.

[0167] In certain embodiments, the therapeutically effective amount ofthe peptide fragment is provided by providing to the animal arecombinant nucleic acid having a nucleotide sequence encoding thepeptide fragment or a biologically active analog thereof, and which iscapable of expressing the peptide fragment or biologically active analogthereof in vivo. The peptide fragment is administered to the animal byadministering the recombinant nucleic acid. The nucleic acid can be,e.g., any of the polynucleotides described herein. Certain preferrednucleic acids are polynucleotides encoding the peptide comprising theamino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 or biologically active analogs thereof, e.g., thesequences as set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 andSEQ ID NO:39. Other preferred nucleic acids are polynucleotides encodingthe peptide comprising the amino acid sequence as set forth in SEQ IDNO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or biologicallyactive analogs thereof, e.g., the sequences as set forth in SEQ IDNO:110, SEQ ID NO:11, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 andSEQ ID NO:115. In certain embodiments, the recombinant nucleic acid is agene therapy vector, e.g., as described herein.

[0168] In certain embodiments, the therapeutically effective amount ofthe peptide fragment is provided by providing to the animal acomposition comprising animal cells wherein a recombinant nucleic acidhaving a nucleotide sequence encoding the peptide fragment has beenintroduced ex vivo into the animal cells so as to express the peptidefragment in the animal cells. The peptide fragment is administered tothe animal by administering the animal cells having the recombinantnucleic acid. Preferably, the recombinant nucleic acid is a gene therapyvector, e.g., as described herein. Preferably, the animal cells arederived from the animal to be treated or allogeneic cells.

[0169] Administration of an agent, e.g., a peptide or nucleic acid canbe accomplished by any method which allows the agent to reach the targetcells. These methods include, e.g., injection, deposition, implantation,suppositories, oral ingestion, inhalation, topical administration, orany other method of administration where access to the target cells bythe agent is obtained. Injections can be, e.g., intravenous,intradermal, subcutaneous, intramuscular or intraperitoneal.Implantation includes inserting implantable drug delivery systems, e.g.,microspheres, hydrogels, polymeric reservoirs, cholesterol matrices,polymeric systems, e.g., matrix erosion and/or diffusion systems andnon-polymeric systems, e.g., compressed, fused or partially fusedpellets. Suppositories include glycerin suppositories. Oral ingestiondoses can be enterically coated. Inhalation includes administering theagent with an aerosol in an inhalator, either alone or attached to acarrier that can be absorbed. The agent can be suspended in liquid,e.g., in dissolved or colloidal form. The liquid can be a solvent,partial solvent or non-solvent. In many cases, water or an organicliquid can be used.

[0170] In certain embodiments of the invention, the administration canbe designed so as to result in sequential exposures to the agent oversome time period, e.g., hours, days, weeks, months or years. This can beaccomplished by repeated administrations of the agent, e.g., by one ofthe methods described above, or alternatively, by a controlled releasedelivery system in which the agent is delivered to the animal over aprolonged period without repeated administrations. By a controlledrelease delivery system is meant that total release of the agent doesnot occur immediately upon administration, but rather is delayed forsome time. Release can occur in bursts or it can occur gradually andcontinuously. Administration of such a system can be, e.g., by longacting oral dosage forms, bolus injections, transdermal patches orsubcutaneous implants. Examples of systems in which release occurs inbursts include, e.g., systems in which the agent is entrapped inliposomes which are encapsulated in a polymer matrix, the liposomesbeing sensitive to a specific stimulus, e.g., temperature, pH, light,magnetic field, or a degrading enzyme, and systems in which the agent isencapsulated by an ionically-coated microcapsule with a microcapsulecore-degrading enzyme. Examples of systems in which release of the agentis gradual and continuous include, e.g., erosional systems in which theagent is contained in a form within a matrix, and diffusional systems inwhich the agent permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be, e.g., in the form of pellets orcapsules.

[0171] The agent can be administered prior to or subsequent to theappearance of disease symptoms. In certain embodiments, the agent isadministered to patients with familial histories of the disease, or whohave phenotypes that may indicate a predisposition to the disease, orwho have been diagnosed as having a genotype which predisposes thepatient to the disease, or who have other risk factors.

[0172] The agent is administered to the animal in a therapeuticallyeffective amount. By therapeutically effective amount is meant thatamount which is capable of at least partially preventing or reversingthe disease. A therapeutically effective amount can be determined on anindividual basis and will be based, at least in part, on considerationof the species of animal, the animal's size, the animal's age, theefficacy of the particular agent used, the longevity of the particularagent used, the type of delivery system used, the time of administrationrelative to the onset of disease symptoms, and whether a single,multiple, or controlled release dose regimen is employed. Atherapeutically effective amount can be determined by one of ordinaryskill in the art employing such factors and using no more than routineexperimentation.

[0173] In certain preferred embodiments, the concentration of the agentif it is a peptide is at a dose of about 0.1 to about 1000 mg/kg bodyweight/day, more preferably at about 0.1 to about 500 mg/kg/day, morepreferably yet at about 0.1 to about 100 mg/kg/day, and most preferablyat about 0.1 to about 5 mg/kg/day. Preferably, the dosage form is suchthat it does not substantially deleteriously affect the animal.

[0174] In certain embodiments, a therapeutically effective amount of anagent which is a peptide can be administered by providing to the animala nucleic acid encoding the peptide and expressing the peptide in vivo.Preferably, the dosage form is such that it does not substantiallydeleteriously affect the animal. Nucleic acids encoding the peptide canbe administered in any biologically effective carrier, e.g. anyformulation or composition capable of effectively delivering thenucleotide sequence for the peptide to cells in vivo. Approachesinclude, e.g., insertion of the nucleic acid into viral vectors,including, e.g., retrovirus, adenovirus, adeno-associated virus, herpesvirus and Semliki Forest virus vectors. Viral vectors can be deliveredto the cells, e.g., by infection or transduction using the virus. Viralvectors can also be delivered to the cells, e.g., by physical means,e.g., by electroporation, lipids, cationic lipids, liposomes, DNA gun,Ca₃(PO₄)₂ precipitation, or delivery of naked DNA. In certain preferredembodiments, the virus is administered by injection, e.g., intramuscularinjection, in a dose range of about 10³ to about 10¹⁰ infectiousparticles per injection per treatment, more preferably in a dose rangeof about 10⁵ to about 10⁸ infectious particles per injection pertreatment. Single or multiple doses can be administered over a givenperiod of time, depending, e.g., upon the disease. An alternative isinsertion of the nucleic acid encoding the peptide into a bacterial oreukaryotic plasmid. Plasmid DNA can be delivered to cells with the helpof, e.g., cationic liposomes (lipofectin™; Life Technologies, Inc.,Gaithersburg, Md.) or derivatized (e.g., antibody conjugated) polylysineconjugates, gramicidin S, streptolysin O artificial viral envelopes orother such carriers or delivery aids, as well as direct injection of thegene construct or Ca₃(PO₄)₂ precipitation carried out in vivo, or by useof a gene gun. The above-described methods are known to those skilled inthe art and can be performed without undue experimentation.

[0175] In certain embodiments, the nucleic acid is administered to theanimal by introducing ex vivo the nucleic acid into cells of the animalor allogeneic cells, and administering the cells having the nucleic acidto the animal. Any cell type can be used. In certain embodiments, thecells having the introduced nucleic acid are expanded and/or selectedafter the nucleic acid transfer. The cells having the transferrednucleic acid are subsequently administered to the animal. Preferably,the cells are administered to the animal in a dose range of about 1×10⁶to about 1×10⁹ cells/dosage/treatment, and most preferably at about1×10⁷ to about 1×10⁸ cells/dosage/treatment. The cells can beadministered by any method which results in delivering the transferrednucleic acid in the cells to the desired target. For example, the cellscan be implanted directly into a specific tissue of the animal, orimplanted after encapsulation within an artificial polymer matrix.Examples of sites of implantation include the lungs or airways, skin,conjunctiva, central nervous system, peripheral nerve, a grafted kidney,or an inflamed joint.

[0176] Choice of the particular delivery system will depend on suchfactors as the intended target and the route of administration, e.g.,locally or systemically. Targets for delivery of the agent can be, e.g.,specific target cells which are causing or contributing to disease. Forexample, the target can be resident or infiltrating cells in the lungsor airways that are contributing to an asthmatic illness; resident orinfiltrating cells in the nervous system that are contributing todemyelinating disease; resident or infiltrating cells responsible forrejection of a kidney graft; grafted cells whose activation producesgraft-versus-host disease; or resident or infiltrating cells whoseactivation underlies inflammation or arthritic degeneration of a joint.Administration can be directed to one or more cell types, and to one ormore subsets of cells within a cell type, so as to be therapeuticallyeffective, by methods known to those skilled in the art. For example,the agent can be coupled to an antibody, to a ligand to a cell surfacereceptor, or to a toxin component, or can be contained in a particlewhich is selectively internalized into cells, e.g., liposomes, or avirus where the viral receptor binds specifically to a certain celltype, or a viral particle lacking the viral nucleic acid, or can beadministered by local injection. In certain embodiments, administrationis done in a prenatal animal or embryonic cell.

[0177] In other embodiments, the agent is a non-peptide molecule, e.g.,an organic or inorganic compound, e.g., as isolated from a library oforganic or inorganic compounds as described herein. Exemplary doses ofsuch agents include milligram or microgram amounts of the compound perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram). It is furthermoreunderstood that appropriate doses of such compounds depend upon thepotency of the compound with respect to the activity to be modulated.When one or more of these compounds is to be administered to an animal(e.g., a human) in order to inhibit the protein-protein interactionbetween calcineurin and NFAT, e.g., to modulate an immune response or totreat a disease or condition described herein, a physician,veterinarian, or researcher may, for example, prescribe a relatively lowdose at first, subsequently increasing the dose until an appropriateresponse is obtained. In addition, it is understood that the specificdose level for any particular animal subject will depend upon a varietyof factors including the activity of the specific compound employed, theage, body weight, general health, gender, and diet of the subject, thetime of administration, the route of administration, the rate ofexcretion, any drug combination, and the degree of expression oractivity to be modulated.

[0178] In certain embodiments, other therapy is additionallyadministered. For example, another therapeutic agent, chemotherapy,radiation or surgery, is additionally administered to the animal, eithersimultaneously or at different times.

[0179] The invention also includes a method for treating a diseaseinvolving hyperactivity or inappropriate activity of the immune system,a transplant graft rejection, or graft-versus-host disease in an animal.An animal in need of treatment for a disease involving hyperactivity orinappropriate activity of the immune system, a transplant graftrejection, or graft-versus-host disease, is provided. A therapeuticallyeffective amount of an agent, e.g., a peptide fragment of the conservedregulatory domain of NFAT protein or a biologically active analogthereof, or a non-peptide agent, e.g., an organic or inorganic compound,capable of inhibiting protein-protein interaction between calcineurinand NFAT, is provided. The agent, e.g., peptide fragment or biologicallyactive analog thereof, or non-peptide agent is administered to theanimal in a therapeutically effective amount such that treatment of thedisease involving hyperactivity or inappropriate activity of the immunesystem, transplant graft rejection, or graft-versus-host disease,occurs.

[0180] Immune system diseases involving hyperactivity or inappropriateactivity of the immune system include, e.g., acute immune diseases,chronic immune diseases and autoimmune diseases. Examples of suchdiseases include rheumatoid arthritis, inflammatory bowel disease,allogeneic or xenogeneic transplantation rejection (organ, bone marrow,stem cells, other cells and tissues), graft-versus-host disease,aplastic anemia, psoriasis, lupus erytematosus, inflammatory disease,type I diabetes, asthma, pulmonary fibrosis, scleroderma,dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome,Kawasaki disease, Hashimoto's thyroiditis, Graves' disease, myastheniagravis, pemphigus vulgaris, autoimmune hemolytic anemia, idiopathicthrombopenia, chronic glomerulonephritis, Goodpasture's syndrome,Wegner's granulomatosis, multiple sclerosis, cystic fibrosis, chronicrelapsing hepatitis, primary biliary cirrhosis, uveitis, allergicrhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's disease,ulcerative colitis, Guilllain-Barre syndrome, chronic inflammatorydemyelinating polyradiculoneuropathy, eczema and autoimmune thyroiditis.Transplant graft rejections can result from tissue or organ transplants.Graft-versus-host disease can result from bone marrow or stem celltransplantation.

[0181] The methods of the present invention can also be utilized totreat conditions and diseases that are not immune mediated, but whichnevertheless involve the protein-protein interaction between calcineurinand NFAT. Examples include myocardial hypertrophy, dilatedcardiomyopathy, excessive or pathological bone resorption, excessiveadipocyte differentiation, obesity, and reactivation of latent humanherpesvirus-8 or other viruses.

[0182] Treating is meant to include, e.g., preventing, treating,reducing the symptoms of, or curing the disease or condition. In certainembodiments, the therapeutically effective amount of the peptidefragment or biologically active analog thereof is administered byproviding to the animal a nucleic acid encoding the peptide fragment orbiologically active analog thereof, and expressing the peptide fragmentor biologically active analog thereof in vivo.

[0183] The invention also includes a method for treating an animal atrisk for a disease involving hyperactivity or inappropriate activity ofthe immune system, a transplant graft rejection, or graft-versus-hostdisease. An animal at risk for a disease involving hyperactivity orinappropriate activity of the immune system, a transplant graftrejection, or graft-versus-host disease, is provided. A therapeuticallyeffective amount of a peptide fragment of the conserved regulatorydomain of NFAT protein capable of inhibiting protein-protein interactionbetween calcineurin and NFAT, or a biologically active analog thereof,is provided. The peptide fragment or biologically active analog thereofis administered in a therapeutically effective amount such thattreatment occurs.

[0184] Being at risk for the disease can result from, e.g., a familyhistory of the disease, a genotype which predisposes to the disease, orphenotypic symptoms which predispose to the disease.

[0185] The invention also includes a method for gene therapy. An animalcell is genetically modified such that it is able to express a peptidefragment or biologically active analog thereof of the conservedregulatory domain of NFAT protein, the peptide fragment being capable ofinhibiting calcineurin-mediated NFAT activation, so as to effect genetherapy. In certain embodiments, the animal cells are geneticallymodified by introducing into the cells a recombinant nucleic acid havinga nucleotide sequence encoding the peptide fragment and which is capableof expressing the peptide fragment in vivo. Preferably, the recombinantnucleic acid is a gene therapy vector, e.g., as described herein.

[0186] The invention also includes a pharmaceutical composition fortreating an immune-related disease or condition in an animal comprisinga therapeutically effective amount of a peptide fragment of theconserved regulatory domain of NFAT protein capable of inhibitingprotein-protein interaction between calcineurin and NFAT, or abiologically active analog thereof, and a pharmaceutically acceptablecarrier. The peptide fragment can be, e.g., any of the peptide fragmentsdescribed herein. Pharmaceutically acceptable carriers include, e.g.,water, saline, dextrose, glycerol, ethanol, liposomes and lipidemulsions.

[0187] The invention also includes a pharmaceutical composition fortreating an immune-related disease or condition in an animal, comprisinga therapeutically effective amount of a recombinant nucleic acid havinga nucleotide sequence encoding a peptide fragment of the conservedregulatory domain of NFAT protein capable of inhibiting protein-proteininteraction between calcineurin and NFAT, or a biologically activeanalog thereof, and a pharmaceutically acceptable carrier. The nucleicacid can be, e.g., any of the polynucleotides described herein.

[0188] The invention also includes a pharmaceutical composition fortreating an immune-related disease or condition in an animal, comprisinga therapeutically effective amount of animal cells wherein a recombinantnucleic acid having a nucleotide sequence encoding a peptide fragment ofthe conserved regulatory domain of NFAT protein capable of inhibitingprotein-protein interaction between calcineurin and NFAT, or abiologically active analog thereof, has been introduced into the animalcells so as to express the peptide fragment; and a pharmaceuticallyacceptable carrier. Preferably, the animal cells are cells derived fromthe animal to be treated or allogeneic cells.

[0189] The invention also includes a method for inhibitingprotein-protein interaction between calcineurin and NFAT in vivo. A cellhaving calcineurin and NFAT is provided. A peptide fragment or abiologically active analog thereof of the conserved regulatory domain ofNFAT protein capable of inhibiting protein-protein interaction betweencalcineurin and NFAT is provided. The calcineurin and peptide fragmentor biologically active analog thereof are contacted in vivo such thatprotein-protein interaction between the calcineurin and the NFAT isinhibited.

[0190] The invention also includes a method for inhibitingprotein-protein interaction between calcineurin and NFAT in vitro.Calcineurin and NFAT are provided. A peptide fragment or a biologicallyactive analog thereof of the conserved regulatory domain of NFAT proteincapable of inhibiting protein-protein interaction between calcineurinand NFAT is provided. The calcineurin and peptide fragment orbiologically active analog thereof are contacted b vitro such thatprotein-protein interaction between the calcineurin and the NFAT isinhibited.

[0191] The invention also includes a method for evaluating an agent foruse in modulating an immune response. A cell is provided. An agent,e.g., a peptide fragment of the conserved regulatory domain of NFATprotein or biologically active analogs thereof, is provided. The effectof the agent on an aspect of calcineurinmediated NFAT activation isevaluated. A change in the aspect of calcineurin-mediated NFATactivation is indicative of the usefulness of the agent in modulating animmune response.

[0192] Any aspect of calcineurin-mediated NFAT activation can beevaluated, e.g., protein-protein interaction between calcineurin andNFAT, dephosphorylation of NFAT by calcineurin, recruitment of NFAT tothe nucleus in a cell, conformational change in NFAT, or activation ofNFAT-dependent gene transcription.

[0193] The invention also includes a method for high throughputscreening of candidate agents to identify an agent that inhibitsprotein-protein interaction between calcineurin and NFAT. A firstcompound is provided. The first compound is calcineurin or abiologically active derivative thereof, or the first compound is NFAT ora biologically active derivative thereof. A second compound is providedwhich is different from the first compound and which is labeled. Thesecond compound is calcineurin or a biologically active derivativethereof, or the second compound is NFAT or a biologically activederivative thereof. A candidate agent is provided. The first compound,second compound and candidate agent are contacted with each other. Theamount of label bound to the first compound is determined. A reductionin protein-protein interaction between the first compound and the secondcompound as assessed by label bound is indicative of the usefulness ofthe agent in inhibiting protein-protein interaction between calcineurinand NFAT. Preferably, the reduction is assessed relative to the samereaction without addition of the candidate agent.

[0194] In certain embodiments, the first compound which is provided isattached to a solid support. Solid supports include, e.g., resins, e.g.,agarose and a multiwell plate. In certain embodiments, the methodincludes a washing step after the contacting step, so as to separatebound and unbound label.

[0195] By high-throughput screening is meant that the method can be usedto screen a large number of candidate agents easily and quickly. Inpreferred embodiments, a plurality of candidate agents are contactedwith the first compound and second compound. The different candidateagents can be contacted with the other compounds in groups orseparately. Preferably, each of the candidate agents is contacted withboth the first compound and the second compound in separate wells. Forexample, the method can screen libraries of potential agents. Librariesare meant to include, e.g., natural product libraries, organic chemicallibraries, combinatorial chemical libraries, peptide libraries, andmodified peptide libraries, including, e.g., D-amino acids,unconventional amino acids, or N-substituted amino acids. Preferably,the libraries are in a form compatible with screening in multiwellplates, e.g., 96-well plates. The assay is particularly useful forautomated execution in a multiwell format in which many of the steps arecontrolled by computer and carried out by robotic equipment. Thelibraries can also be used in other formats, e.g., synthetic chemicallibraries affixed to a solid support and available for release intomicrodroplets.

[0196] Calcineurin and biologically active derivatives thereof is meantto include, e.g., intact calcineurin; calcineurin A chain; fragments ofcalcineurin that are biologically active in binding NFAT, e.g., acatalytic domain fragment of the calcineurin A chain that binds to NFAT;analogs of calcineurin or a calcineurin fragment that are biologicallyactive in binding NFAT; and chimeric recombinant proteins, e.g.,calcineurin or a biologically active fragment of calcineurin fused toanother peptide or protein such that calcineurin retains itsNFAT-binding activity. The calcineurin and its biologically activederivatives can be natural, recombinant or synthesized. In certainpreferred embodiments, the calcineurin can be from, e.g., a mammal,e.g., a human, or yeast. Calcineurin can be obtained, e.g., in cellextracts of cells that normally express calcineurin, or by expressingrecombinant calcineurin protein in eukaryotic or prokaryotic cells. Incertain embodiments, calmodulin is included in the assay so as to confercalcium responsiveness on calcineurin.

[0197] NFAT and biologically active derivatives thereof is meant toinclude intact NFAT, e.g., NFAT1, NFAT2, NFAT3 or NFAT4; fragments ofNFAT that are biologically active, e.g., that retain the ability to forma protein-protein interaction with calcineurin or the ability ofinhibiting the binding of NFAT to calcineurin, e.g., a peptide fragmentof the conserved regulatory domain of NFAT, as described herein; analogsof NFAT or an NFAT fragment that are biologically active; and chimericrecombinant proteins, e.g., NFAT or a biologically active fragment ofNFAT fused to another peptide or protein such that NFAT retains itsactivity. Examples of such chimeric recombinant proteins include: (i)NFAT fused to maltose-binding protein or glutathione S-transferase (GST)so as to immobilize the NFAT on a solid support, e.g., a resin; (ii)NFAT fused to green fluorescent protein or one of its variants for usein a fluorescence assay or a fluorescence energy transfer assay; and(iii) NFAT fused to a peptide tag so as to allow its recognition by aspecific antibody or its labeling by a specific protein kinase. The NFATand its biologically active derivatives can be natural, recombinant orsynthesized. NFAT can be, e.g., a mammalian protein, e.g., human ormurine. NFAT can be obtained, e.g., in cell extracts of cells thatnormally express NFAT, or by expressing a recombinant NFAT protein ineukaryotic or prokaryotic cells.

[0198] In certain embodiments, the NFAT derivative is a mutated NFATthat has increased affinity for calcineurin. Such mutants are obtained,e.g., by applying a two-hybrid screen to mutagenized NFAT (see, e.g.,Mendelsohn and Brent, Curr. Opin. Biotech. 5:482-486 (1994); Goldfarb etal, J Biol Chem 271:2683-2688 (1996); Colas et al, Nature 380:548-550(1996)) or using any other selection or screening method known to thoseskilled in the art, or produced by introducing into NFAT amino acidsubstitutions identified through screening peptide libraries or phagedisplay libraries (see, e.g., Kast and Hilvert, Curr. Opin. Struct.Biol. 7:470-479 (1997)). Advantages of using mutant NFAT proteins thatbind calcineurin with higher affinity are reducing the amount ofradiolabeled calcineurin required for an assay, permitting morestringent washing, and expanding the range of assays that produce adetectable signal. A compelling example is Example 17, wherein the highsensitivity and high signal-to-noise ratio of the screening assay isdirectly attributable to utilization of the optimized NFAT derivative,VIVIT peptide.

[0199] In certain embodiments, the first compound is calcineurin or abiologically active derivative thereof, and the second compound is NFATor a biologically active derivative thereof. In other embodiments, thefirst compound is NFAT or a biologically active derivative thereof, andthe second compound is calcineurin or a biologically active derivativethereof. The solid support to which the first compound is attachedincludes, e.g., Sepharose beads, SPA beads and a multiwell plate.Preferably, SPA beads (microspheres that incorporate a scintillant) areused when the assay is performed without a washing step, e.g., in ascintillation proximity assay. Preferably, Sepharose beads are used whenthe assay is performed with a washing step. The second compound can belabeled with any label that will allow its detection, e.g., aradiolabel, a fluorescent agent, biotin, a peptide tag, or an enzymefragment. Preferably, the second compound is radiolabeled, e.g., with¹²⁵I or ³H.

[0200] In certain embodiments, the enzymatic activity of an enzymechemically conjugated to, or expressed as a fusion protein with, thefirst or second compound, is used to detect bound protein. A bindingassay in which a standard immunological method is used to detect boundprotein is also included. Methods based on surface plasmon resonance,as, e.g., in the BIAcore biosensor (Pharmacia Biosensor, Uppsala,Sweden) or evanescent wave excitation of fluorescence are particularlysuited to measure recruitment of, e.g., NFAT (or fluorescently labeledNFAT) to a surface on which calcineurin is immobilized. In certain otherembodiments, the interaction of NFAT and calcineurin is detected byfluorescence resonance energy transfer (FRET) between a donorfluorophore covalently linked to NFAT (e.g., a fluorescent groupchemically conjugated to NFAT, or a variant of green fluorescent protein(GFP) expressed as an NFAT-GFP chimeric protein) and an acceptorfluorophore covalently linked to calcineurin, where there is suitableoverlap of the donor emission spectrum and the acceptor excitationspectrum to give efficient nonradiative energy transfer when thefluorophores are brought into close proximity through theprotein-protein interaction of NFAT and calcineurin.

[0201] In certain embodiments, the protein-protein interaction isdetected by reconstituting domains of an enzyme, e.g., pgalactosidase(see Rossi et al, Proc. Natl. Acad. Sci. USA 94:8405-8410 (1997)). Thedetection method used is appropriate for the particular enzymaticreaction, e.g., by chemiluminescence with Galacton Plus substrate fromthe Galacto-Light Plus assay kit (Tropix, Bedford, Mass.) or byfluorescence with fluorescein di-β-D-galactopyranoside (MolecularProbes, Eugene, Oreg.) for β-galactosidase. Competition of theprotein-protein interaction by the candidate agents or by the 13-mer or26-mer inhibitory peptides described herein, is evident in a reductionof the measured enzyme activity. This assay can be performed withproteins in vitro or in vivo. An advantage of this embodiment in vivo isthat only compounds sufficiently permeable through the membrane ofliving cells will be scored positive, and thus agents most likely toreach effective concentrations within cells when administeredtherapeutically can be identified. Measurement of reconstitutedS-galactosidase activity in living cells has been demonstrated withfluorescein di-β-D-galactopyranoside (Molecular Probes, Eugene, Oreg.)as substrate. See Rossi et al., Proc. Natl. Acad. Sci. USA 94:8405-8410(1997).

[0202] In certain embodiments, the protein-protein interaction isassessed by fluorescence ratio imaging (Bacskai et al, Science260:222-226 (1993)) of suitable chimeric constructs of NFAT andcalcineurin in cells, or by variants of the two-hybrid assay (Fearon etal, Proc Natl Acad Sci USA 89:7958-7962 (1992); Takacs et al, Proc NatlAcad Sci USA 90:10375-10379 (1993); Vidal et al, Proc Natl Acad Sci USA93:10315-10320 (1996); Vidal et al, Proc Natl Acad Sci USA93:10321-10326 (1996)) employing suitable constructs of NFAT andcalcineurin and tailored for a highthroughput assay to detect compoundsthat inhibit the NFAT calcineurin interaction. These embodiments havethe advantage that the cell permeability of compounds that act asspecific inhibitors in the assay is assured.

[0203] Any false positives identified in these assays, such as proteindenaturants or natural product samples contaminated with a proteaseactivity, can be detected and eliminated through secondary assays thatdemonstrate that their inhibitory action is nonspecific, e.g., that suchcompounds interfere with known protein-protein interactions betweenpairs of proteins unrelated to NFAT and calcineurin.

[0204] The invention also includes a method for high-throughputscreening of candidate agents to identify an agent that inhibitsdephosphorylation of NFAT by calcineurin. Phosphorylated NFAT isprovided. Calcineurin or a biologically active derivative thereof havingenzymatic activity is provided. A candidate agent is provided. Thephosphorylated NFAT, the calcineurin or biologically active derivativethereof, and the candidate agent are contacted with each other inreaction media, e.g., buffer, under conditions that allow enzymaticactivity of calcineurin. In certain embodiments, the NFAT is separatedfrom the reaction media. It is determined whether phosphate remainsassociated with the NFAT. If phosphate remains associated is indicativeof the usefulness of the agent in dephosphorylation of NFAT bycalcineurin.

[0205] In certain embodiments, the phosphorylated NFAT is labeled. Thephosphate can be labeled with any label that will allow its detection.Preferably, the phosphate is radiolabeled, e.g., with ³²P or ³³P. Incertain embodiments, determination of whether phosphate remainsassociated with the NFAT is accomplished by determining the release oflabeled phosphate in the reaction media, or the retention of labeledphosphate on the NFAT. A reduction in release of labeled phosphate fromthe NFAT by the calcineurin, or an increase in retention of labeledphosphate on the NFAT, is indicative of the usefulness of the agent ininhibiting dephosphorylation of NFAT by calcineurin. Preferably, thereduction is assessed relative to the same reaction without addition ofthe candidate agent.

[0206] In certain embodiments, the phosphorylated NFAT that is providedis attached to a solid support.

[0207] In preferred embodiments, a plurality of candidate agents arecontacted with the phosphorylated NFAT which optionally is attached to asolid support, and the calcineurin or biologically active derivativethereof. The different candidate agents can be contacted with the NFATand calcineurin in groups or separately. Preferably, each of thecandidate agents is contacted with the NFAT and the calcineurin inseparate wells.

[0208] NFAT in its phosphorylated form can be obtained by any methodknown to those skilled in the art. Methods that involve enzymaticlabeling using a protein kinase in vitro are preferred where ³²P isincorporated, since high specific activities can be achieved. ERK2phosphorylates GST-NFAT1 fusion protein on sites accessible tocalcineurin. A combination of protein kinase A (protein kinase,catalytic subunit; Sigma Chemical Co., St. Louis, Mo.) and glycogensynthase kinase 3β (New England Biolabs, Beverly, Mass.) phosphorylatesGST-NFAT2 on a set of sites that correspond to those phosphorylated bvivo (Beals et al, Science 275:1930-1933 (1997)). For assays thatmeasure ³²P-phosphate remaining covalently associated with the proteinafter the incubation with calcineurin, the background signal due tophosphate incorporated at calcineurin-insensitive sites may be loweredby preblocking all substrate sites for the kinase in a reaction withunlabeled-ATP, treating with calcineurin, washing, and thenincorporating ³²P in a second kinase reaction that labels predominantlythose sites that are accessible to calcineurin.

[0209] For assays that use nonradioactive phosphorylated NFAT or ³²Plabeled NFAT in native form, mammalian cells or insect cells expressinghigh levels of recombinant protein after transformation with abaculovirus vector can be used to obtain sufficient NFAT inphosphorylated form. A method for preparation of fully phosphorylatednative NFAT1 from mammalian cells is described in Shaw et al., Proc.Natl. Acad. Sci. USA 92:11205-11209 (1995). Fully phosphorylated NFAT1can also be obtained by lysis of the cells in a detergent-containingbuffer, provided that sufficient concentrations of phosphataseinhibitors, e.g., 60 mM sodium pyrophosphate, 10 mM EDTA, and 5 mM EGTA,are included in the lysis buffer. Since the inhibitors are subsequentlywashed away after NFAT is purified away from endogenous phosphatases,their inclusion at the lysis step does not compromise a subsequentenzymatic assay using calcineurin. Minor modifications of theseprocedures that may be necessary for isolation of phosphorylated NFATfrom insect cells include, e.g., use of additional protease inhibitors,additional phosphatase inhibitors, or higher concentrations of theinhibitors. The NFAT expression construct introduced into these cells ina baculovirus vector preferably encodes a chimeric protein including anepitope tag or hexahistidine tag, or a fusion protein withglutathione-S-transferase, or some similar fusion protein providing forfacile purification of the expressed protein. In some cases,phosphorylated NFAT or ³²P-labeled NFAT can be obtained by coexpressionof NFAT and a constitutively active kinase in bacteria, e.g., in E.coli.

[0210] In certain embodiments, determining dephosphorylation of NFAT canbe accomplished by examining specific sites remaining phosphorylated inthe NFAT protein after treatment with calcineurin. A compound is scoredas positive if it increases the retention of covalently bound phosphateon a specific site or sites of NFAT. Preferably, the presence or absenceof covalently bound phosphate is determined using antibodies, or afunctionally equivalent reagent, e.g., genetically engineeredantibodies, minibodies or aptamers, that discriminate betweenphosphorylated and unphosphorylated forms of a specific peptide in thecontext of the larger protein or protein fragment. NFAT peptides thatcan be used include, e.g., FQNIPAHYSPRT (SEQ ID NO:94), PAHYSPRTSPIM(SEQ ID NO:95), or SPRTSPIMSPRT (SEQ ID NO:96)(from the sequenceFQNIPAHYSPRTSPIMSPRT (SEQ ID NO:97), residues 207 to 226 in murineNFATI) or PVPRPASRSSSP (SEQ ID NO:98), RPASRSSSPG (SEQ ID NO:99), orASRSSSPGAKRR (SEQ ID NO:100)(from the sequence PVPRPASRSSSPGAKRR (SEQ IDNO:101), residues 239 to 255 in murine NFAT1). Antibodies tophosphorylated or dephosphorylated NFAT peptides can be raised, e.g., byimmunization of rabbits. See e.g., Czernik et al, Methods Enzymol201:264-283 (1991) for preparation and characterization of serum ormonoclonal antibodies using short synthetic peptides (10-12 residues)corresponding to the sequence surrounding a phosphorylation site. Theunphosphorylated peptides can be obtained by conventional methods ofchemical synthesis, e.g., Merrifield solid phase synthesis. Thephosphopeptides can be obtained, e.g., by in vitro phosphorylation ofthe synthetic peptides with kinase in instances where the syntheticpeptide includes flanking residues that form a consensus site for the,kinase (Czernik et al, Methods Enzymol 201:264-283 (1991)), or, e.g., bychemical synthesis of peptides phosphorylated on serine or threonineresidues (Perich J W, Methods Enzymol 201:225-233 (1991)). The antiseraor monoclonal antibodies can be tested to determine whether they showthe ability to discriminate between phosphorylated and unphosphorylatedpeptides, e.g., by dot immunoblotting or by ELISA (Czemik et al, MethodsEnzymol 201:264-283 (1991)). To ensure that a specific antiserum ormonoclonal antibody reagent discriminates between phosphopeptide anddephosphopeptide in the context of NFAT protein, and to select ahigh-affinity reagent with low background signal in the high-throughputscreening assay, the candidate antiserum or monoclonal antibody can befurther tested under the conditions to be used in the high-throughputscreening assay.

[0211] Any antibody based assay can be used. Preferably, an automatedassay that reflects the relative amount of phosphorylated orunphosphorylated peptide is used. For example, a very efficient methodof monitoring dephosphorylation is to use fluorescence resonance energytransfer between two appropriately labeled antibodies to two distinctphosphopeptides, capable of simultaneous binding to the protein, whichare added directly to the reaction after stopping the phosphataseincubation with, e.g., EGTA, another inhibitor of calcineurin activity,or by mild protein denaturation. Variants of this embodiment include,e.g., antibodies directed against the dephosphorylated forms of twodistinct NFAT peptides, corresponding miniaturized antibodies(“minibodies”; Tramontano et al, J. Mol. Recognit. 7:9-24 (1994); Martinet al, EMBO J. 13:5303-5309 (1994); Martin et al, J. Mol Biol 255:86-97(1996)), or peptide aptamers (Colas et al, Nature 380:548-550 (1996))selected to recognize phosphorylated or dephosphorylated forms of NFATpeptides. In some variants of this embodiment, a single fluorescentlylabeled antibody, minibody, or peptide aptamer that binds to aphosphorylated or dephosphorylated form of an NFAT peptide is pairedwith fluorescently tagged NFAT in a fluorescence resonance energytransfer assay; or a fluorescently labeled antibody, minibody, orpeptide aptamer directed to a phosphopeptide or dephosphopeptide ispaired in a fluorescence resonance energy transfer assay with a secondlabeled antibody, minibody, or peptide aptamer that binds constitutivelyto NFAT or a peptide tag at a site unaffected by phosphorylation ordephosphorylation of the protein. In embodiments in which theantibodies, minibodies, or peptide aptamers are continuously presentduring the incubation with calcineurin, the reagents preferably aredirected against the dephosphopeptide so that they will not interferewith access of calcineurin to the phosphopeptide.

[0212] In certain embodiments, the screening assay uses measurements ofrelease of ³²P from a reporter site introduced into recombinant NFAT, ormeasurements with antibodies to the phosphorylated or dephosphorylatedforms of a reporter site introduced into recombinant NFAT. The insertedreporter site takes the form of a short peptide sequence, known to be anefficient substrate for a specific protein kinase, that is geneticallyengineered into NFAT. The inserted site that is used is able to bephosphorylated efficiently in vitro in its context within the NFATprotein, the phosphorylated site is dephosphorylated by calcineurin, andthe efficiency of dephosphorylation is reduced by a 13-mer or 25-merinhibitory peptide described herein, showing that the specificprotein-protein recognition of NFAT by calcineurin is essential fordephosphorylation.

[0213] In certain embodiments, the interaction of NFAT with the enzymeactive site of calcineurin, as distinct from the recognition site wherethe protein-protein interaction is disrupted by a 13-mer or 25-merinhibitory peptide described herein, is assessed by examining theactivity of calcineurin against a second substrate in the presence ofNFAT. Because binding of NFAT to the recognition site brings substratepeptides within NFAT into proximity of the active site, and indeed intothe active site as evidenced by their consequent dephosphorylation, NFATexhibits competition with other substrates that are dephosphorylated bycalcineurin. In the absence of binding to the recognition site, NFAT maystill compete with other substrates, but only at significantly higherconcentrations. Since the agents sought in this assay, like the 13-merpeptide, do not inhibit calcineurin activity against substrates otherthan NFAT, their presence in the assay will reduce competition by NFATand cause an apparent stimulation of calcineurin activity against theassayed substrate.

[0214] Such an assay uses a standard calcineurin phosphatase assay. Theconcentration of NFAT required for competition depends on many factors,e.g., the substrate, assay time, temperature and assay buffer, which aredetermined for particular reaction conditions by simple testing of arange of NFAT concentrations in pilot experiments. The control reactionthat shows the dependence of the competition on NFAT-calcineurinrecognition is carried out with inclusion of a 13-mer or 25-merinhibitory peptide described herein. In one embodiment, the measurementof calcineurin phosphatase activity is made by determining the releaseof ³²P from a phosphopeptide substrate, e.g., ³²P-RII peptide. Inanother embodiment, the enzymatic activity of calcineurin is determinedby use of a biotinylated phosphopeptide substrate that can be captured,subsequent to the incubation with calcineurin, on a streptavidin-coatedsolid support and probed with an antibody specific for thedephosphopeptide. In certain embodiments, detection is by formation of afluorescent product with, e.g., 4-nitrophenylphosphate (MolecularProbes, Eugene, Oreg.) or fluorescein diphosphate (Molecular Probes,Eugene, Oreg.) as substrate. Those skilled in the art are aware of manyalternative ways to assess the enzymatic activity of calcineurin. Anadvantage of this embodiment is that such assays do not requirephosphorylated NFAT.

[0215] In any of the dephosphorylation assays described herein, agentsthat inhibit NFAT dephosphorylation by preventing the specificinteraction between calcineurin and NFAT are identified, as well asagents that act by a different mechanism, e.g., as general inhibitors ofcalcineurin. The latter general inhibitors can be eliminated, e.g., by asecond screening assay which tests the agent's ability to inhibitdephosphorylation of other known substrates of calcineurin. The assaybased on competition with a second substrate may identify generalactivators of calcineurin, which can likewise be eliminated, e.g., in asecond screening assay that tests the agent's ability to augmentdephosphorylation of the second substrate when the incubation isperformed in the absence of NFAT. Further, to confirm that the mechanismof action is interference with the protein-protein interaction of NFATand calcineurin, all compounds identified in the dephosphorylation assaycan, e.g., be tested directly for their interference in theprotein-protein interaction of NFAT and calcineurin using the assaysdescribed herein.

[0216] The invention also includes a method for high-throughputscreening of candidate agents to identify an agent that inhibitsconformational change in NFAT from dephosphorylation by calcineurin.Phosphorylated NFAT is provided. In certain embodiments, thephosphorylated NFAT is attached to a solid support. Calcineurin or abiologically active derivative thereof having enzymatic activity isprovided. A candidate agent is provided. An oligonucleotide having anNFAT site is provided. The phosphorylated NFAT, calcineurin orbiologically active derivative thereof, and the candidate agent arecontacted with each other in reaction media under conditions that allowenzymatic activity of calcineurin. Specific binding of NFAT to theoligonucleotide having the NFAT site is determined. A reduction ofbinding is indicative of the usefulness of the agent in inhibitingconformational change in NFAT from dephosphorylation by calcineurin.Preferably, the reduction is assessed relative to the same reactionwithout addition of the candidate agent.

[0217] NFAT changes its conformation as a direct consequence ofdephosphorylation by calcineurin in such a way as to dramaticallyincrease its specific binding to DNA (Park et al, J. Biol Chem270:20653-20659 (1995); Shaw et al, Proc Natl Acad Sci USA92:11205-11209 (1995)). Specific binding of NFAT to DNA can be simplyassessed in an assay that is suitable for high throughput screening.Thus, this alteration in DNA binding can be used to detectdephosphorylation of NFAT and to screen for compounds that are capableof inhibiting the dephosphorylation. NFAT in phosphorylated form,obtained as described above, is treated with calcineurin in the presenceof a candidate agent to be tested. Preferably, control samples ofphosphorylated NFAT are incubated (i) in the absence of calcineurin,(ii) in the presence of calcineurin with no added candidate agent, and(iii) in the presence of calcineurin and known inhibitors, e.g., the13-mer peptide or 25-mer peptide as described herein, or CsA/cyclophilincomplexes. At the end of the incubation, specific binding of NFAT to anoligonucleotide, e.g., a double-stranded oligonucleotide, e.g., DNA,incorporating an NFAT site, e.g., the distal NFAT site of the murineIL-2 promoter (Jain et al, Nature 356:801-804 (1992)) or the P1 site ofthe murine IL-4 promoter (Rooney et al, Immunity 2:473-483 (1995)), ismeasured. In one embodiment, this measurement is made by incubating thesample with biotinyl-DNA, incorporating the NFAT binding site, thenfurther incubating with streptavidin-SPA beads and ¹²⁵I-labeled antibodyagainst NFAT. In this embodiment, scintillation counting of ¹²⁵I labelgives a measure of the NFAT-DNA complex that has formed. Compounds ofinterest in the assay are those that prevent or inhibit the increase inDNA binding that results from incubation of phosphorylated NFAT withcalcineurin. In embodiments in which the compounds tested are notseparated from NFAT before the DNA binding step, preferably, it isfurther shown that a compound of interest does not directly inhibit theability of NFAT to bind to DNA, e.g., by examining DNA binding in thesame assay when the test compound is added only after the incubationwith calcineurin is completed, or by examining the effect of thecompound on the binding of bacterially-expressed NFAT. As is known toone skilled in the art, there are many effectively equivalent methodsfor measuring the binding of NFAT to DNA including, e.g., recruitment of³H-DNA to NFAT bound via anti-67.1 antiserum (Ho et al, J Biol Chem269:28181-28186 (1994)) on protein A-SPA beads, competition by unlabeledNFAT with a fixed amount of ¹²⁵I-NFAT for binding to biotinyl-DNAimmobilized on streptavidin-SPA beads, inclusion in the DNA bindingreaction of c-Fos and c-Jun proteins to increase the affinity of theinteraction, and using another solid phase support.

[0218] In an alternative, the conformational change, and thereforedephosphorylation, may be detected directly by using a probe thatrecognizes specifically a region or determinant of NFAT that is exposedonly after dephosphorylation. An example is the nuclear localizationsequence (NLS) of NFAT, which is masked until dephosphorylation, butthen becomes accessible for binding of other proteins, e.g., theimportin proteins that direct dephosphorylated NFAT to the nucleus incells. Exposure of the NLS, or of a tag peptide introduced intorecombinant NFAT in place of the NLS, may be detected, e.g., in animmunoassay with an appropriate antibody. In another alternative, theconformational change in NFAT may be detected by fluorescence resonanceenergy transfer (FRET) using a recombinant NFAT protein labeled withappropriate fluorophores at two distinct sites, as has been illustratedfor calmodulin (Miyawaki et al, Nature 388:883-887 (1997)), or by FRETbetween fluorophore-labeled minibodies directed to distinct sites on thesurface of NFAT whose relative position changes as a result of theconformational change, or by alteration in the intrinsic fluorescence ofNFAT upon dephosphorylation.

[0219] The invention also includes a method for high-throughputscreening of candidate agents to identify an agent that inhibitscalcineurin-dependent import of NFAT into the nucleus of a cell. Cellsexpressing NFAT are provided. A stimulant that activates NFAT throughthe calcium/calcineurin pathway is provided. A candidate agent isprovided. The cells, stimulant and candidate agent are contacted witheach other. The presence or absence of nuclear NFAT in the cells isdetermined. A reduction in nuclear NFAT is indicative of the agentinhibiting calcineurin-dependent import of NFAT into the nucleus of acell. Preferably, the reduction is assessed relative to the samereaction without addition of the candidate agent.

[0220] This assay is based on the calcineurin-dependent difference inlocalization of NFAT in unstimulated and stimulated cells. Cellsexpressing NFAT, e.g., endogenous or recombinant NFAT, are incubated inthe presence of a stimulant, e.g., calcium ionophore, aneurotransmitter, or a biologically active peptide, known to triggeractivation of NFAT via the calcium/calcineurin pathway (for examples,see Table 1 in Rao et al, Annu. Rev. Immunol. 15:707-747 (1997)).Preferably, control samples of cells are incubated without addition ofthe stimulant, or in the presence of the stimulant and with knowninhibitors of calcineurin-dependent NFAT activation, e.g., CsA andFK506. Determining the presence or absence of nuclear NFAT, and also,preferably cytoplasmic NFAT, can be accomplished by any method known toone skilled in the art. Preferably, localization to the nucleus insamples incubated in the presence of the candidate agent is comparedwith that of control samples incubated with the stimulant only. Agentsare scored as positive if they interfere with the calcineurin-dependentimport of NFAT to the cell nucleus.

[0221] Examples of determining the localization of NFAT include fixingthe cells after the contacting step with histological fixative andexamining by microscopy or other means capable of detecting thedifference between cells having cytoplasmic NFAT and cells havingnuclear NFAT. In some embodiments, NFAT is detected byimmunocytochemical staining. In some embodiments, the localization of anNFAT fusion protein, e.g., NFAT-GFP, is detected by fluorescencemicroscopy. Localization of NFAT to the nucleus can be scored, e.g., bymicroscopy using visual inspection. In some embodiments, visualinspection is aided by automation, or the localization of NFAT isdetermined in an automated assay. In certain embodiments of an automatedassay, cells are stained with, e.g., two distinct fluorophores, a firstfluorophore that detects NFAT (e.g., fluorescently labeled NFAT orfluorescent antibodies that bind to NFAT) and a second fluorophore thatlabels cell nuclei. In these embodiments, the nuclear localization ofNFAT is quantitated by assessing colocalization of the two labels, e.g.,the average level of NFAT label is determined in pixels that shownuclear labeling above a designated threshold level that is easilydetermined by examining the positive and negative control samples. Inyet other embodiments, line or raster scanning is used to excitefluorescence, or a method sensitive to the spatial frequency of thefluorescent signal is employed.

[0222] As with other assays based on detecting the calcineurin-NFATinteraction or its consequences in cells, assays based on thecalcineurin-dependent import of NFAT into the cell nucleus have theadvantage that the cell permeability of specific inhibitors identifiedin the assay is assured.

[0223] In some embodiments, these cell-based assays are used to screen,e.g., a retroviral expression library, or other peptide or proteinexpression libraries, for those recombinant proteins capable ofinterfering with the calcineurin-mediated activation of NFAT.

[0224] The cellular assay examining cytoplasmic or nuclear localizationof NFAT is also useful as a diagnostic test to confirm normalphysiological function in cells derived from an animal, e.g., human, todetect or classify pathological or abnormal function of immune systemcells, or to identify stimuli or sources of activation of immune systemcells. For example, an immune disorder can be detected or classified bydocumenting abnormal activation (constitutively nuclear NFAT protein) ina class of cells, or by documenting an abnormal failure to translocateNFAT in response to a stimulus. In another example, the source of anallergic response can be determined, e.g., by testing candidateallergens for their ability to induce nuclear translocation of NFAT inan indicator mast cell line stably expressing NFAT1(1-460)-GFP, wherethe indicator mast cells have first been exposed to IgE from an animalor a human, and where further exposure to an effective allergen willtherefore cause activation of the cells through their Fc receptors andnuclear import of NFAT-GFP.

[0225] The invention includes a method for assessing the state of NFATactivation of immune system cells from an animal. Immune system cellsisolated from an animal are provided. The presence or absence of nuclearNFAT in the cells is determined. The presence of nuclear NFAT in thecells is indicative of activation of NFAT in the cells. The cells can beisolated by any method known to those skilled in the art, e.g., bybiopsy or aspiration. In certain embodiments, the cells are infiltratingcells at a site of inflammation or in a tumor. Preferably, the presenceor absence of nuclear NFAT is determined by histological staining, e.g.,immunocytochemical staining, of the cells.

[0226] The invention also includes a method for assessing the ability ofimmune system cells isolated from an animal to respond to an NFATactivating signal. Immune system cells from an animal are provided, thecells being unactivated for NFAT. A stimulant that activates NFAT isprovided. The cells are contacted with the stimulant. The presence orabsence of nuclear NFAT in the cells is determined. A reduction innuclear NFAT is indicative of impairment of the ability of the cells torespond to an NFAT activating signal. Preferably, the reduction isassesssed relative to cells isolated from a normal animal. This assaycan be used, e.g., to monitor the level of immune function in certainimmunocompromised patients.

[0227] The invention also includes a method for identifying a stimulantthat can activate NFAT in immune system cells isolated from an animal.Immune system cells isolated from an animal are provided. A candidatestimulant is provided. The cells are contacted with the candidatestimulant. The presence or absence of nuclear NFAT in the cells isdetermined. The presence of nuclear NFAT is indicative of the stimulantactivating NFAT in the cells. In preferred embodiments, the stimulant isan allergen.

[0228] By allergen is meant an agent that elicits IgE-mediatedreactions. This assay can be used, e.g., to monitor unrespon-siveness toa pathogen or tolerance to a specific antigen.

[0229] The invention also includes a method for identifying an allergen.An animal cell line expressing NFAT is provided. IgE from an animal,e.g., a human, is provided. A candidate allergen is provided. The cellline is contacted with the IgE. The cell line is contacted with thecandidate allergen. Preferably, the cell line is contacted with thecandidate allergen after the cell line is contacted with the IgE. Thepresence or absence of nuclear NFAT in cells of the cell line isdetermined. The presence of nuclear NFAT is indicative of the candidateallergen being an allergen.

[0230] Small Molecules for Inhibition of the Protein-Protein Interactionof Calcineurin and NFAT

[0231] As discussed above, the invention includes methods for highthroughput screening of candidate agents, e.g., agents that areinitially members of an organic chemical library, to identify agentsthat inhibit the protein-protein interaction between calcineurin andNFAT. The present invention also includes small organic molecules, e.g.,nonpeptide molecules, that have been isolated using the methodsdescribed herein. It is contemplated that persons skilled in the art canreadily modify the organic molecules specifically described herein to,e.g., obtain a molecule optimized for administration to an animal.

[0232] In general, organic molecules useful for the present inventionhave a molecular weight of less than 2500 Daltons (Da). The smallmolecules can be, e.g., from at least about 100 Da to about 2000 Da(e.g., between about 100 to about 2000 Da, about 100 to about 1750 Da,about 100 to about 1500 Da, about 100 to about 1250 Da, about 100 toabout 1000 Da, about 100 to about 750 Da, about 100 to about 500 Da,about 200 to about 1500, about 500 to about 1000, about 300 to about1000 Da, or about 100 to about 250 Da).

[0233] Organic molecules useful for the present invention can exhibitthe ability to interact with, e.g., bind to, calcineurin or a fragmentthereof, with an affinity constant of at least about 2×10⁴M⁻¹, e.g.,least about 10⁵ M⁻¹, at least about 10⁶ M⁻¹, at least about 10⁷ M⁻¹, orat least about 10⁸ M⁻¹, or stronger. In view of the common docking siteon calcineurin for all NFAT-family proteins, inhibitors that bind tocalcineurin can interfere with activation of all NFAT proteins, and canprovide the beneficial effects of some currently used immunosuppressantdrugs, e.g., CsA and FK506, potentially without undesirable side effectscaused by such drugs.

[0234] Organic molecules useful for the present invention can exhibitthe ability to interact with, e.g., bind to, NFAT or a fragment thereof,with an affinity constant of at least about 2×10⁴ M⁻¹, e.g., at leastabout 10⁶ M⁻¹ at least about 10⁷ M⁻¹, or at least about 10⁸ M⁻¹.Inhibitors of this class can be complementary to a surface of NFAT,rather than to the NFAT-docking site on calcineurin. The primarystructure of NFAT proteins is only partially conserved in the domainthat interacts with calcineurin. Hence, in some cases, such smallmolecules of the present invention can bind preferentially to oneNFAT-family protein and/or to a subset of NFAT-family proteins, overothers. Such a characteristic is desirable in treatment of certaindisorders of the immune system, in preventing or treating myocardialdisease without compromising immune function, and in treating conditionswherein pathological signalling by NFAT is predominantly caused by onetype of NFAT protein or by a definable subset of NFAT proteins. Further,the conformation of NFAT is altered by dephosphorylation. Such smallmolecules can display a preference for binding to phosphorylated(inactive) NFAT or to dephosphorylated (activated) NFAT, making thesmall molecule capable of reducing calcineurin activity more effectivelyagainst one than the other, and preferentially modulating either therate of activation of phosphorylated NFAT or the rate of inactivation ofdephosphorylated NFAT. Such preferential binding could result in, forexample, a selective attenuation of strong rapid transcriptionalsignalling via the calcineurin-NFAT pathway, with a lesser effect onweak sustained signalling, hence selectively reducing the expression ofa subgroup of NFAT target genes or altering the time course ofexpression of NFAT target genes in a beneficial way.

[0235] Exemplary organic molecules of the present invention areillustrated below as formulas (I), (IV) and (III), and exemplarysubstitutions are given in accompanying Tables 2, 3, and 4. TABLE 2

Compound R₁ R₂ R₃ R₄ R₅ INCA1 H Ph Cl CHAc₂ O INCA1A Me OEt Br H NMeINCA1B H Ph Br H O INCA1C Me OEt Br H O INCA1D H 4-MePh Cl Cl O INCA1E H4-MePh Br H O

[0236] TABLE 3

Cmpd R₁ R₂ R₃ R₄ R₅ R₆ R₇ INCA2 Cl Cl — H H Double Bond INCA2A Cl Cl — HMe Double Bond INCA2B Cl H — H Me Double Bond INCA2C Br H — H Me DoubleBond INCA2D Cl Cl — H Me Cl Cl INCA2E H H — H H Double Bond INCA2F Cl H— H Cl Double Bond INCA2G Br H — H Cl Double Bond INCA2H Cl Cl OMe H MeDouble Bond INCA2I Cl Cl OMe H Cl Double Bond INCA2J H H — Me Me DoubleBond INCA2K Cl Cl OBu H Me Double Bond INCA2L DDC H — H Me Double BondINCA2M NMe₂ H — H H Double Bond

[0237] TABLE 4

Cmpd. R₁ R₂ R₃ R₄ R₅ R₆ R₇ INCA6 O H H O H H H

[0238] Other representative inhibitory compounds containing scaffoldsother than those of formulae (I), (II) and (III) are included inAppendix I, and are enumerated as Compounds INCA3, CAN4, INCA5, INCA7,INCA8, INCA9, INCA10, CAN33, CAN11, CAN30, CAN13, CAN22, CAN14, CAN15,CAN21, CAN16, CAN17, and CAN19.

[0239] As used herein, the term “halo” or “halogen” refers to anyradical of fluorine, chlorine, bromine or iodine. The term “alkyl”refers to a hydrocarbon chain that may be a straight chain or branchedchain, containing the indicated number of carbon atoms. For example,C₁-C₁₀ indicates that the group may have from 1 to 10 (inclusive) carbonatoms in it. The term “lower alkyl” refers to a C₁-C₈ alkyl chain. Theterm “alkoxy” refers to an —O-alkyl radical. The term “alkylene” refersto a divalent alkyl (i.e., —R—). The term “alkylenedioxo” refers to adivalent species of the structure —O—R—O—, in which R represents analkylene. The term “aminoalkyl” refers to an alkyl substituted with anamino. The term “mercapto” refers to an —SH radical. The term“thioalkoxy” refers to an —S-alkyl radical.

[0240] The term “aryl” refers to a 6-carbon monocyclic or 10-carbonbicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of eachring may be substituted by a substituent. Examples of aryl groupsinclude phenyl, naphthyl and the like. The term “arylalkyl” or the term“aralkyl” refers to alkyl substituted with an aryl. The term“arylalkoxy” refers to an alkoxy substituted with aryl.

[0241] The term “cycloalkyl” includes saturated and partiallyunsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably3 to 8 carbons, and more preferably 3 to 6 carbons, wherein thecycloalkyl group additionally may be optionally substituted. Preferredcycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

[0242] The term “heteroaryl” refers to an aromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.Examples of heteroaryl groups include pyridyl, furyl or furanyl,imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl,quinolinyl, indolyl, thiazolyl, and the like. The termn“heteroarylalkyl” or the term “heteroaralkyl” refers to an alkylsubstituted with a heteroaryl. The term “heteroarylalkoxy” refers to analkoxy substituted with heteroaryl.

[0243] The term “heterocyclyl” refers to a nonaromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein0, 1, 2 or 3 atoms of each ring may be substituted by a substituent.Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl,dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

[0244] The term “oxo” refers to an oxygen atom, which forms a carbonylwhen attached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

[0245] The term “substituents” refers to a group “substituted” on analkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atomof that group. Suitable substituents include, without limitation, halo,hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl,alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl,alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl,acyloxy, cyano, and ureido groups. In one aspect, the substituents areindependently selected from the group consisting of C₁-C₆ alkyl, C₃-Cgcycloalkyl, (C₁-C₆)alkyl(C₃-C₈)cycloalkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl,cyano, amino, C₁-C₆alkylamino, di(C₁-C₆)alkylamino, benzylamino,dibenzylamino, nitro, carboxy, carbo(C₁-C₆)alkoxy, trifluoromethyl,halogen, C₁-C₆ alkoxy, C₆-C₁₀ aryl, (C₆-C₁₀)aryl(C₁-C₆)alkyl,(C₆-C₁₀)aryl(C₁-C₆)alkoxy, hydroxy, C₁-C₆ alkylthio, C₁-C₆alkylsulfinyl, C₁-C₆ alkylsulfonyl, C₆-C₁₀ arylthio, C₆-C₁₀arylsulfinyl, C₆-C₁₀ arylsulfonyl, C₆-C₁₀ aryl,(C₁-C₆)alkyl(C₆-C₁₀)aryl, and halo(C₆-C₁₀)aryl.

[0246] Combinations of substituents and variables envisioned by thisinvention are only those that result in the formation of stablecompounds. The term “stable,” as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and which maintainsthe integrity of the compound for a sufficient period of time to beuseful for the purposes detailed herein (e.g., therapeutic orprophylactic administration to a subject).

[0247] As can be appreciated by the skilled artisan, further methods ofsynthesizing the compounds of the formulae herein will be evident tothose of ordinary skill in the art. Additionally, the various syntheticsteps may be performed in an alternate sequence or order to give thedesired compounds. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagentsfor OrganicSynthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

[0248] The compounds useful in the present invention can also beobtained as part of a library of organic compounds. Combinatorialtechniques suitable for synthesizing the compounds described herein areknown in the art as exemplified by Obrecht, D. and Villalgrodo, J. M.,Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries, Pergamon-Elsevier ScienceLimited (1998), and include those such as the “split and pool” or“parallel” synthesis techniques, solid-phase and solution-phasetechniques, and encoding techniques (see, for example, Czarnik, A. W.,Curr. Opin. Chem. Bio., (1997) 1, 60.

[0249] In an alternate embodiment, the compounds described herein may beused as platforms or scaffolds that may be utilized in combinatorialchemistry techniques for preparation of derivatives of the organiccompounds described herein and/or chemical libraries of compounds. Suchderivatives and libraries of compounds have biological activity and areuseful for identifying and designing compounds that inhibit theprotein-protein interaction of calcineurin and NFAT.

[0250] Thus, one embodiment relates to a method of using the compoundsdescribed in the formulae herein for generating derivatives or chemicallibraries comprising: 1) providing a body comprising a plurality ofwells; 2) providing one or more compounds of the formulae describedherein in each well; 3) providing an additional one or more chemicals ineach well; 4) isolating the resulting one or more products from eachwell. An alternate embodiment relates to a method of using the compoundsdescribed in the formulae herein for generating derivatives or chemicallibraries comprising: 1) providing one or more compounds of the formulaedescribed herein attached to a solid support; 2) treating the one ormore compounds of the formulae described herein attached to a solidsupport with one or more additional chemicals; 3) isolating theresulting one or more products from the solid support. In the methodsdescribed above, “tags” or identifier or labeling moieties may beattached to and/or detached from the compounds of the formulae herein ortheir derivatives, to facilitate tracking, identification or isolationof the desired products or their intermediates. Such moieties are knownin the art. The chemicals used in the aforementioned methods mayinclude, for example, solvents, reagents, catalysts, protecting groupand deprotecting group reagents and the like. Examples of such chemicalsare those that appear in the various synthetic and protecting groupchemistry texts and treatises referenced herein.

[0251] The compounds of this invention may contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are expresslyincluded in the present invention. The compounds of this invention mayalso be represented in multiple tautomeric forms, in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein (e.g., alkylation of a ring system may result inalkylation at multiple sites, the invention expressly includes all suchreaction products). All such isomeric forms of such compounds areexpressly included in the present invention. All crystal forms of thecompounds described herein are expressly included in the presentinvention.

[0252] As used herein, the compounds of this invention, including thecompounds of formulae described herein, are defined to includepharmaceutically acceptable derivatives or prodrugs thereof. A“pharmaceutically acceptable derivative or prodrug” means anypharmaceutically acceptable salt, ester, salt of an ester, or otherderivative of a compound of this invention which, upon administration toa recipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored derivatives and prodrugs arethose that increase the bioavailability of the compounds of thisinvention when such compounds are administered to a mammal (e.g., byallowing an orally administered compound to be more readily absorbedinto the blood) or which enhance delivery of the parent compound to abiological compartment (e.g., the brain or lymphatic system) relative tothe parent species. Preferred prodrugs include derivatives where a groupwhich enhances aqueous solubility or active transport through the gutmembrane is appended to the structure of formulae described herein, orto derivatives thereof.

[0253] The compounds of this invention may be modified by appendingappropriate functionalities to enhance selective biological properties.Such modifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

[0254] Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from pharmaceutically acceptableinorganic and organic acids and bases. Examples of suitable acid saltsinclude acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate,fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate,persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, salicylate, succinate, sulfate, tartrate, thiocyanate,tosylate and undecanoate. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts. Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts. This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

[0255] The organic molecules described herein, or derivatives thereof,can be combined with a pharmaceutically acceptable carrier to create apharmaceutical composition. The pharmaceutically acceptable carrier canbe any solvent, dispersion medium, coating, antibacterial and antifungalagent, isotonic and absorption delaying agent, and any of the like thatare physiologically compatible. The carrier can be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the organic molecule may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound. The compositions ofthis invention may be in a variety of forms. These include, for example,liquid, semi-solid and solid dosage forms, such as liquid solutions(e.g., injectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. The preferred formdepends on the intended mode of administration and therapeuticapplication.

[0256] The following non-limiting examples further illustrate thepresent invention.

EXAMPLES Example 1 The SPRIEITPS Sequence of NFAT1 is Involved inNuclear Import of NFAT1

[0257] This example illustrates that mutations in the SPRIEITPS (SEQ IDNO:17) sequence in the conserved motif-2 (CM2) region (amino acidresidues 110-118) of the NFAT1 conserved regulatory domain (amino acidresidues 100-397) inhibit translocation of the mutant NFAT1 protein fromthe cytoplasm to the nucleus.

[0258] A triple CM2 mutant was generated which disrupts the sequence¹¹⁰SPRIEITPS¹¹⁸ (SEQ ID NO:17) of NFAT1 by replacing each of the threeamino acid residues Arg¹¹² (R¹¹²), Glu¹¹⁴(E¹¹⁴), and Thr¹¹⁶(T¹¹⁶), withan alanine residue. These mutations were generated following theprocedures described in Kunkel et al., Methods Enzymol. 154:367-382(1987). The mutant proteins were expressed with an HA epitope tag inC1.7W2 murine T cells and in HeLa cells, and analyzed for nucleartranslocation in response to ionomycin stimulation. The mutant proteinsbehaved identically in both cell types. Translocation of wild type andmutant NFAT1 to the nucleus was measured by immunocytochemistry asfollows. HeLa cells expressing HA-tagged full length wild type NFAT1 ormutant NFAT1 were left unstimulated or activated with ionomycin (3 μM,10 min). NFAT1 was detected with mouse anti-HA antibody (12CA5) and Cy3™goat anti-mouse IgG, and visualized using a rhodamine filter set on aZeiss Axioskop microscope at a magnification of 630×. Results indicatedthat the triple CM2 mutation impaired translocation of NFAT1 to thenucleus upon ionomycin stimulation. Wild type NFAT1, as well as themutant ST21, having two mutations in this same region, in which the twoserine residues flanking the SPRIEITPS (SEQ ID NO:17) motif weresubstituted by alanine residues, showed normal translocation.

[0259] The triple CM2 mutations also impaired nuclear translocation of aGFP fusion protein containing only the N-terminal domain of NFAT1 (aminoacids 1-460), NFAT1(1-460)-GFP. 24 h after transfection with a plasmidencoding wild type or mutant NFAT-GFP, the HeLa cells or C1.7W2 murine Tcells were left untreated or stimulated with ionomycin (2 μM, 10 min).Nuclear translocation of NFAT1 was visualized by fluorescencemicroscopy. This impairment of nuclear translocation indicated that theSPRIEITPS (SEQ ID NO:17) motif is involved in the nuclear import ofNFAT1, and that the effect of the CM2 mutation does not require anintact DNA binding domain (amino acid residues 398-680 of murine NFAT1)or the C-terminal domain of NFAT1 (amino acid residues 681-923, 681-927and 681-1064 in the three known isoforms of murine NFAT1).

[0260] The role in nuclear translocation of each of the amino acidresidues, R¹¹², E¹¹⁴ and T¹¹⁶, was also assessed individually. Thesingle amino acid mutation T¹¹⁶ to A, in the SPRIEITPS (SEQ ID NO:17)sequence of NFAT1, inhibited translocation in response to ionomycinstimulation almost to the same extent as did the triple CM2 mutation.Single mutations of R¹¹² and E¹¹⁴ also impaired translocation, but to alesser extent than the triple CM2 mutation.

Example 2 The SPRIEITPS Sequence of NFAT1 is Required for EffectiveDephosphorylation by Calcineurin

[0261] This example illustrates that mutations in the SPRIEITPS (SEQ IDNO:17) sequence of NFAT1 inhibit dephosphorylation of the mutant NFAT1protein by calcineurin.

[0262] The inability of the CM2 mutant, containing the three mutationsdescribed in Example 1, to translocate to the nucleus correlated withits very limited dephosphorylation in stimulated cells. When transientlyexpressed either in HeLa cells or in C1.7W2 murine T cells, wild typeNFAT1(1-460)-GFP fusion protein was dephosphorylated in response toionomycin stimulation (3 μM, 10 min) as assessed by its shift inmigration on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The morecomplete shift observed in C1.7W2 T cells under these conditionsreflects the higher level of calcineurin activity in this cell line. Incontrast, the triple CM2 mutant NFAT1 (1-460)-GFP showed no change inmigration after ionomycin stimulation of HeLa cells, and only a slightshift after stimulation of C1.7W2 T cells, indicating thatdephosphorylation was inhibited.

[0263] The difference in ionomycin sensitivity between HA-tagged wildtype NFAT1 (1-460)-GFP and the corresponding triple CM2 mutant wastested in C1.7W2 T cells stimulated with a range of ionomycinconcentrations (0.07 to 6 μM) for 10 min. Significant dephosphorylationof HA-tagged wild type NFAT1 was achieved in cells stimulated with 220nM ionomycin, the dephosphorylation being complete with 660 nMionomycin. In contrast, dephosphorylation of the CM2 mutant protein wasincomplete even in cells stimulated with 6 μM ionomycin, a concentration10 times higher than that required to induce complete dephosphorylationof wild type NFAT1. Dephosphorylation of both wild type NFAT1 and theCM2 mutant was inhibited by cyclosporin A (CsA)(500 nM), indicating thatthe dephosphorylation remained calcineurindependent. Immunocytochemicalexperiments confirmed that the CM2 mutant did not translocate to thenucleus even in C1.7W2 cells stimulated with 6 μM ionomycin.

[0264] The triple CM2 mutant also displayed significantly reducedsensitivity to treatment with exogenous calcineurin in vitro.Cytoplasmic extracts from HeLa cells expressing HA-tagged wild typeNFAT1 or the triple CM2 mutant NFAT1 protein were incubated withcalcineurin (200 nM-2.5 μM) and calmodulin for 30 min at 30° C. Thesamples were then resolved by SDS-PAGE and the phosphorylation state wasanalyzed by Western blotting (Shaw et al., Proc Natl Acad Sci USA92:11205-11209 (1995); Luo et al., Proc Natl Acad Sci USA 93:8907-8912(1996)) with anti-HA antibody. Dephosphorylation of wild type NFAT1 wasapparent with 200 nM calcineurin, whereas the triple CM2 mutant wasmarkedly less sensitive, with only partial dephosphorylation occurringin the presence of 2.5 μM calcineurin.

[0265] The role in NFAT1 dephosphorylation of each of the amino acidresidues, R¹¹², E¹¹⁴ and T¹¹⁶, was also assessed individually by Westernblotting. The single amino acid substitution T¹¹⁶ to A, in the SPRIEITPS(SEQ ID NO:17) sequence of NFAT1, inhibited dephosphorylation inresponse to ionomycin stimulation almost to the same extent as did thetriple CM2 mutation. Single mutations of R¹¹² and E¹¹⁴ also impaireddephosphorylation, but to a lesser extent than the triple CM2 mutation.

Example 3 Peptides Spanning the SPRIRITPS Sequence of NFAT1 Interferewith Recognition and Dephosphorylation of NFAT1 by Calcineurin

[0266] This example illustrates that peptides spanning the SPRIEITPS(SEQ ID NO:17) sequence of NFAT1 interfere with recognition anddephosphorylation of NFAT1 by calcineurin.

[0267] The results from Examples 1 and 2 indicated that the CM2mutations in the SPRIEITPS (SEQ ID NO:17) sequence impaired the abilityof calcineurin to recognize NFAT1 as a substrate and to dephosphorylateit, either in vivo or in cell extracts. One possibility was that theSPRIEITPS (SEQ ID NO:17) sequence represented a region ofNFAT-calcineurin contact and that mutation of this sequence impaired thetargeting of calcineurin to NFAT.

[0268] To test whether the SPRIEITPS (SEQ ID NO:17) motif was directlyinvolved in the interaction of NFAT with calcineurin, peptides spanningthe CM2 motif of wild type and mutant NFAT1 were tested in anNFAT-calcineurin binding assay for their capacity to block calcineurinbinding to NFAT1. Two wild type peptides were synthesized which spannedthe SPRIEITPS (SEQ ID NO:17) motif, one containing 13 amino acidresidues of murine NFAT1 (SEQ ID NO:22) and one containing 25 amino acidresidues of NFAT1 (SEQ ID NO:29). In the case of the longer peptide, atyrosine residue not present in the NFAT1 sequence was appended at the Cterminus to facilitate chemical coupling to a carrier protein forproduction of antisera, and the peptide tested in this and subsequentexamples (Examples 4 and 5 infra) was therefore a 26-mer. Acorresponding 26-mer peptide incorporating the R¹¹² to A, E¹¹⁴ to A, andT¹¹⁶ to A substitutions of the triple CM2 mutant NFAT1, and with theappended tyrosine residue, was synthesized for use as a control. It isevident from the results below, specifically from the identical effectsof the 13-mer peptide and the 26-mer peptide on calcineurin binding andfrom the lack of effect of the 26-mer mutant peptide on calcineurinbinding, that the C-terminal tyrosine residue has no role in inhibitingthe protein-protein interaction.

[0269]¹²⁵I-labelled calcineurin (14 nM, −7×10⁵ cpm) was incubated for 30min at 4° C. with GST-tagged NFAT1 N-terminal domain (GST NFAT1(1-400))that had been immobilized on glutathione-Sepharose™ beads (PharmaciaBiotech, Piscataway, N.J.), in the absence or presence of differentconcentrations (1 μM to 100 μM) of wild type 13-mer peptide, wild type26-mer peptide, or CM2 mutant 26-mer peptide. At the end of theincubation, the glutathione Sepharose beads with bound NFAT1-calcineurincomplexes were washed on 5 μm hydrophilic Durapore PVDF filters(Millipore Corporation, Bedford, Mass.) to remove unbound calcineurin,and NFAT1-bound calcineurin was quantitated using a gamma counter. Bothpeptides incorporating the wild type sequence SPRIEITPS (SEQ ID NO:17)inhibited the binding of calcineurin to NFAT1. The IC₅₀ for inhibitionby the wild type peptides was very similar, approximately 15 μM for boththe 13-mer and 26-mer. In contrast, the mutated 26-mer peptideincorporating the sequence SPAIAIAPS (SEQ ID NO:82) did not inhibit theNFAT1-calcineurin interaction at concentrations up to 100 μM.

[0270] The ability of the SPRIEITPS peptides to inhibitdephosphorylation of NFAT1 by calcineurin in vitro was measured byWestern blotting analysis. Cytosolic extracts from HeLa cells stablyexpressing HA-tagged N-terminal domain NFAT1(1-460)-GFP were incubatedwith calcineurin (200 nM) and calmodulin (600 nM) in the presence ofdifferent concentrations (16 μM to 2 mM) of wild type 13-mer, wild type26-mer, and CM2 mutant peptides, during 30 min at 30° C. Controls weredone with the calcineurin inhibitors sodium pyrophosphate (10 mM) andcyclosporinA/cyclophilin complexes (15 μM/5 μM). Samples were resolvedby SDS-PAGE and dephosphorylation was assessed with anti-HA antibody12CA5. Both the 13-mer and the 26-mer peptides with wild type sequenceSPRIEITPS (SEQ ID NO:17) inhibited dephosphorylation of NFAT1 atconcentrations in the range from 16 μM to 400 μM, whereas the 26-merpeptide with sequence SPAIAIAPS (SEQ ID NO:82) was only marginallyinhibitory, and only at concentrations above 400 μM. Slightly higherconcentrations of the peptides were required to inhibit the in vitrodephosphorylation than were required to inhibit NFAT-calcineurinbinding, presumably due to some degradation of the peptides by enzymespresent in the cell extracts.

Example 4 The SPRIEITPS Sequence Specifically Targets Calcineurin toNFAT Proteins

[0271] This example illustrates that peptides spanning the SPRIEITPS(SEQ ID NO:17) sequence do not interfere generally with calcineurinphosphatase activity.

[0272] The 13-mer and 26-mer SPRIEITPS peptides were tested to determineif they acted as general inhibitors of calcineurin phosphatase activityby assaying them on the dephosphorylation of a well characterizedcalcineurin substrate, the RII phosphopeptide (Blumenthal et al., J.Biol. Chem. 261:8140-8145 (1986)). ³²P-RII phosphopeptide (100 μM;specific activity 1×10⁹ cpm/μmole) was incubated with calcineurin (100nM) and calmodulin (600 nM). In some samples, FK506/FKBP12 complexes (10μM/10 μM), calcineurin autoinhibitory peptide (30 μM to 400 μM), orSPRIEITPS peptide, 13-mer or 26-mer at different concentrations (30 μMto 400 μM) were preincubated with calcineurin before addition of thecalcineurin mixture to the RII phosphopeptide samples. Thedephosphorylation reaction was allowed to proceed for 30 min at 30° C.after which the reaction was stopped by addition of excess 0.1%trichloroacetic acid, substrate peptide was removed by adsorption onto acation exchange resin (AG50W; BioRad Laboratories, Hercules, Calif.),and released ³²P label in the supernatant was measured in a liquidscintillation counter. Neither of the SPRIEITPS peptides inhibiteddephosphorylation of the RII peptide by calcineurin in the same range ofconcentrations in which they inhibited NFAT1 dephosphorylation. In thesame experiment, calcineurin activity was effectively inhibited by apeptide corresponding to its own autoinhibitory domain and byFK506/FKBP12 complexes.

[0273] Moreover, dephosphorylation of the RII regulatory subunit ofcAMP-dependent protein kinase, a protein substrate of calcineurin(Blumenthal et al., Biol Chem 261:8140-8145 (1986)) from which the RIIphosphopeptide is derived, was not inhibited by a SPRIEITPS peptide fromNFAT1 as shown by an in vitro dephosphorylation assay with recombinantRIIα protein. A 6×Histagged RIIα protein (90 nM), ³²P-labelled in vitroby the PKA catalytic subunit, was incubated with calcineurin (200 nM)and calmodulin (600 nM). In some samples, the calcineurin inhibitorssodium pyrophosphate (20 mM), CsA/cyclophilin complexes (15 μM/5 μM),FK506/FKBP12 complexes (4 μM/4 μM), or different concentrations of the26-mer SPRIEITPS peptide (20 μM to 500 μM) were preincubated withcalcineurin during 20 min on ice before adding ³²P-labelled RIIα proteinto the mixture. Dephosphorylation was allowed to proceed during 45 minat 30° C., samples were resolved by SDS-PAGE, and the gel was stainedwith Coomasie Brilliant Blue, dried, and autoradiographed. Aphosphorimager was used to quantitate the level of ³²P in each lane.Coomasie Brilliant Blue staining showed that equal amounts of thereaction were loaded in each lane. The dephosphorylation of RIIα wasefficiently inhibited by the general phosphatase inhibitor sodiumpyrophosphate and by the calcineurin inhibitors CsA/cyclophilincomplexes and FK506/FKBP12 complexes. In contrast, the 26-mer SPRIEITPSpeptide did not inhibit dephosphorylation when used at theconcentrations (20 μM and 100 μM) required to inhibit NFAT1dephosphorylation, and caused only slight inhibition (18% inhibition) at500 μM.

[0274] Additionally, the 26-mer SPRIEITPS peptide did not inhibit thedephosphorylation of a different protein known to be a calcineurinsubstrate in vivo, the neuronal cytoskeleton protein Tau (Fleming andJohnson, J. Biochem 209:41-47 (1995); Yamamoto et al., J. Biochem118:1224-1231 (1995)). Purified GST-Tau immobilized onglutathione-Sepharose beads was phosphorylated by MAP kinase, washed,and incubated with calcineurin (200 nM) and calmodulin (600 nM) during90 min at 30° C. In some samples, the calcineurin inhibitors sodiumpyrophosphate (10 mM) or CsA/cyclophilin complexes (15 μM/5 μM), or wildtype 26-mer peptide at different concentrations (20 μM to 500 μM) werepreincubated with calcineurin for 20 min on ice before the addition ofTau protein. Samples were resolved by SDS-PAGE and analyzed as above.Dephosphorylation of GST-Tau protein by calcineurin was not inhibited bythe SPRIEITPS peptide at concentrations of 20 μM and 100 μM, and wasminimally inhibited (10%) at 500 μM, a concentration at which thepeptide fully inhibited dephosphorylation of NFAT1. The phosphataseinhibitor sodium pyrophosphate and the calcineurin inhibitor,CsA-cyclophilin complexes, efficiently inhibited dephosphorylation ofthe GST-Tau protein under these conditions.

[0275] In sum, a 13-residue or 26-residue peptide spanning the SPRIEITPS(SEQ ID NO:17) sequence of NFAT1 is a potent inhibitor of theinteraction of NFAT1 with calcineurin, while not affecting either thephosphatase activity of the enzyme or its ability to dephosphorylate theother non-NFAT substrates tested.

Example 5 SPRIEITPS Peptides from NFAT1 Also Interfere with Activationof Other NFAT Family Members

[0276] This example illustrates that peptides spanning the SPRIEITPS(SEQ ID NO:17) sequence from NFAT1 interfere comparably with activationof NFAT2 and NFAT4 despite sequence differences among the NFAT proteins.

[0277] The SPRIEITPS (SEQ ID NO:17) sequence of NFAT1 is highlyconserved in NFAT2, but only partially conserved in NFAT4 which has aCPSIQITSI (SEQ ID NO:20) sequence. NFAT1, NFAT2 and NFAT4 constitute thegroup of NFAT members expressed in immune cells. The inhibitory effectof the SPRIEITPS peptides, 13-mer and 26-mer, of NFAT1 on the binding ofcalcineurin to NFAT1, NFAT2 and NFAT4 was tested. Binding assays of¹²⁵I-calcineurin with GST-tagged N-terminal domains of NFAT1 (residues1-400), NFAT2 (residues 1-418) and NFAT4 (residues 1-400), in thepresence or absence of wild type SPRIEITPS peptides or the mutantcontrol peptide, were done as described above. The results showed thatthe CM2 peptide inhibited the ability of ¹²⁵I-calcineurin to bind to theN-terminal regions of NFAT2 and NFAT4 with a very similar concentrationdependence. The IC₅₀, for inhibition of NFAT2 and NFAT4 calcineurinbinding by the 26-mer peptide was again approximately 15 μM, clearlyindicative of a common calcineurin targeting mechanism involving theSPRIEITPS (SEQ ID NO:17) sequence in NFAT1 and the cognate SPRIEITSC(SEQ ID NO:18) and CPSIQITSI (SEQ ID NO:20) sequences in NFAT2 andNFAT4.

[0278] Similarly, the 13-mer SPRIEITPS peptide from NFAT1 inhibiteddephosphorylation of the N-terminal domain of NFAT4 by calcineurin. TheHA-tagged N-terminal domains of NFAT1 and NFAT4 were expressed as thefusion proteins HA-NFAT1(1-460)-GFP and HA-NFAT4(1-407)-GFP in HEK-293cells. At 24 h after transfection, cytosolic extracts were prepared andequivalent aliquots were incubated without calcineurin or withincreasing concentrations of calcineurin/calmodulin complexes (100 nM to900 nM). Wild type 13-mer peptide of NFAT1 (100 μM or 400 μM finalconcentration in the dephosphorylation reaction) or mutant 26-merpeptide (400 μM final concentration) was added to thecalcineurin/calmodulin preparations 20 min before mixing them with theNFAT-containing cell lysates. Dephosphorylation was allowed to proceedfor 30 min at 30° C., and samples were analyzed by SDS-PAGE and Westernblotting with anti-HA antibody. Lysates from HEK-293 cells expressingHA-NFAT1(1-460)-GFP or HA-NFAT4(1-407)-GFP incubated with calcineurinshowed dephosphorylation of the NFAT proteins at all concentrations ofcalcineurin tested. The addition of wild type 13-mer SPRIEITPS peptide(100 μM or 400 μM) to the assay inhibited dephosphorylation, with moreeffective inhibition in the presence of 400 μM peptide, whereas themutant peptide displayed little or no inhibition even with 400 μMpeptide.

Example 6 An NFAT1 SPRIEITPS 19-mer Peptide Displays ImmunosunpressiveProperties In Vivo

[0279] This example illustrates that expression of a fusion proteinincluding an NFAT1 SPRIEITPS 19-mer peptide inhibits NFAT1 activation invivo as measured by dephosphorylation of NFAT1 in T lymphocytes, nucleartranslocation of NFAT1 in T lymphocytes, and NFAT-mediated geneexpression in T lymphocytes.

[0280] The ability of a GFP-SPRIEITPS-19 fusion protein to inhibitionomycin-induced dephosphorylation of NFAT1 in T lymphocytes was testedas follows. Ionomycin-induced dephosphorylation of HA-taggedNFAT1(1-460)-GFP in T cells expressing the GFP-SPRIEITPS-19 fusionprotein, or expressing related proteins used as controls, was examinedby Western blotting analysis. The GFP-SPRIEITPS-19 expression vector wasmade by introducing a double-stranded oligonucleotide encoding thesequence KPAGASGPSPRIEITPSHEAYD (SEQ ID NO:102) in frame between theBsrGI and NotI sites located 3′ to the green fluorescent protein (GFP)coding sequence in the pEGFP-N1 expression vector (CLONTECH, Palo Alto,Calif.). The codons encoding residues AYD at the C-terminal end of thepeptide sequence were included for convenience in subcloning, and a stopcodon was introduced after the last codon for the peptide sequence. TheGFP-SPAIAIAPS-19 construct was made by subcloning a double-strandedoligonucleotide encoding SGPSPAIAIAPSHEAYD (SEQ ID NO:103) between theBspEI and BsiWI sites in the GFP-SPRIEITPS-19 expression plasmid.Constructs expressing unmodified GFP, GFP-wild type peptide fusionprotein, or GFP-mutant peptide fusion protein, were cotransfectedtogether with expression vector encoding an HA-tagged N-terminal domainof NFAT1 (NFAT1(1-460)-GFP) into C1.7W2 murine T cells. 24 h posttransfection, cells were left untreated or stimulated for 10 min withdifferent concentrations of ionomycin. Whole cell lysates were analyzedfor dephosphorylation of NFAT1 by Western blotting with anti-HA antibodyand for expression of GFP proteins with anti-GFP antibody. The wild typeGFP-SPRIEITPS-19 fusion protein efficiently inhibited ionomycin-induceddephosphorylation of NFAT1, whereas neither GFP alone nor the mutantGFP-SPAIAIAPS-19 protein had any inhibitory effect. Western blottingwith anti-GFP antibody showed that the GFP, GFP-SPRIEITPS-19, andGFP-SPAIAIAPS-19 proteins were expressed at comparable levels. Theinhibition of NFAT1 dephosphorylation by the wild type GFP-SPRIEITPS 19protein was incomplete under strong stimulation conditions. This resultis in agreement with in vitro assays where the concentration ofSPRIEITPS peptide required to inhibit dephosphorylation of NFAT1 andNFAT4 was increasingly higher as the calcineurin concentration in thesamples was increased.

[0281] The ability of GFP-SPRIEITPS-19 fusion protein to inhibit nucleartranslocation of endogenous NFAT1 in T lymphocytes was tested asfollows. C1.7W2 murine T cells expressing GFP, GFP-SPRIEITPS-19 (wildtype peptide), or GFP-SPAIAIAPS-19 (mutant peptide) were stimulated withionomycin (2 μM, 10 min) and processed for immunocytochemistry. NFAT1was visualized with anti-T2B1, a rabbit anti-NFAT1 antiserum directedagainst the C-terminal peptide of NFAT1 isoform C (Wang et al., Ann. NYAcad. Sci. 766:182-194 (1995)) and Cy3TM labeled donkey anti-rabbit IgG(Jackson ImmunoResearch Laboratories, West Grove, Pa.) using a rhodaminefilter set. Simultaneous expression of the GFP constructs in individualcells was assessed by GFP fluorescence using a fluorescein filter set.GFP-SPRIEITPS-19 impaired ionomycin-induced nuclear translocation ofNFAT1, whereas the control proteins, GFP and the mutantGFP-SPAIAIAPS-19, did not. Translocation was completely inhibited,however, only in those cells expressing higher levels of theGFP-SPRIEITPS-19 protein, indicating that, under these conditions ofstrong stimulation, effective inhibition required high intracellularconcentration of the peptide.

[0282] The ability of GFP-SPRIEITPS-19 fusion protein to inhibitNFAT-driven gene transcription in Jurkat T cells was tested as follows.Jurkat human T cells (15×10⁶ cells/transfection) were transfected withexpression plasmids encoding GFP (9 μg plasmid DNA), wild typeGFP-SPRIEITPS-19 fusion protein (variable amounts, supplemented withsufficient plasmid encoding GFP to bring the total plasmid DNA to 9 μg),or mutant GFP-SPAIAIAPS-19 fusion protein, together with an NFAT-drivenluciferase reporter plasmid (2 μg). Aliquots of the transfected cellswere stimulated 24 h after transfection with PMA (20 nM) and ionomycin(1 μM) for 6 hours and luciferase activity in cell lysates was measured.The results showed that GFP-SPRIEITPS-19 inhibited NFAT-mediatedtranscription in a concentration-dependent manner, up to greater than60% inhibition. The mutant GFP-SPAIAIAPS-19 protein was only slightlyinhibitory (−10%), and only at high concentrations. Western blottinganalysis with anti-GFP antibody confirmed that the GFP proteins wereexpressed at equivalent levels, and that the amount of protein expressedwas proportional to the amount of plasmid DNA transfected. Theobservation that the GFP-SPRIEITPS-19 fusion protein did not completelyinhibit NFAT-driven transcription is consistent with the resultsdescribed above, which showed that the inhibitory effect of the proteinwas balanced by the strength of the stimulation conditions, andconsistent with the level of intracellular expression of the GFP-fusionprotein.

[0283] In sum, these results showed that a peptide based on acalcineurin targeting motif of NFAT1 was able to inhibit NFAT1activation and function in vivo.

Example 7 High-Throughput Screen for Inhibitors of Protein-ProteinInteraction Between Calcineurin and NFAT (Using a Washing Step)

[0284] This example illustrates a high-throughput screen for inhibitorsof protein-protein interaction between calcineurin and NFAT, whichutilizes a washing step.

[0285] A fusion protein between glutathione S-transferase and NFAT1,GST-NFAT1(1-400) (Luo et al., Proc Natl Acad Sci USA 93:8907-8912(1996)), is immobilized on glutathione-Sepharose beads (obtained fromPharmacia Biotech, Piscataway, N.J.) by incubation for 30 min at 4° C.in binding buffer (50 mM Tris phosphate pH 8.0, 150 mM NaCl, 5 mM MgCl₂,5 mM 2-mercaptoethanol, 1% Triton X-100, supplemented with 1 mM Naorthovanadate, 20 μM leupeptin, 10 μg/ml aprotinin, 2 mMphenylmethylsulfonyl fluoride). For parallel incubations to monitornonspecific binding (Luo et al., Proc Natl Acad Sci USA 93:8907-8912(1996)), equivalent amounts of GST or GST-LSF, where LSF (Shirra et al.,Mol Cell Biol 14:5076-5087 (1994)) is a DNA-binding protein unrelated toNFAT, are immobilized on glutathione-Sepharose beads. The beads arewashed, resuspended in binding buffer, distributed to multiple wells,and the candidate agents are added. The agents may be supplied as anatural products library, e.g., microbial broths or extracts fromdiverse stains of bacteria, fungi, and actinomycetes (MDS Panlabs,Bothell, Wash.); a combinatorial chemical library, e.g., an Optiverse™Screening Library (MDS Panlabs, Bothell, Wash.); an encodedcombinatorial chemical library synthesized using ECLiPS™ technology(Pharmacopeia, Princeton, N.J.); or another organical chemical,combinatorial chemical, or natural products library assembled accordingto methods known to those skilled in the art and formatted forhigh-throughput screening. Total binding is measured in some samples inthe absence of an added candidate agent. As a positive control forinhibition in the assay, binding is assessed in the presence of aneffective concentration (200 μM) of a 13-mer or 26-mer peptide of theconserved regulatory domain of NFAT protein described herein which iscapable of inhibiting protein-protein interaction between calcineurinand NFAT. The specificity of this positive control inhibition ismonitored in incubations with similar concentrations of a mutant peptidedescribed herein which is inactive in inhibiting such interaction. Eachreaction is supplemented with 100 μM CaCl₂, 800 nM calmodulin (Sigma,St. Louis, Mo.)(to confer calcium responsiveness on calcineurin) and 14nM ¹²⁵I-calcineurin (final concentrations). The incubation is carriedout for 30 min at 4° C. Iodinated calcineurin is prepared by thefollowing procedure. Calcineurin (100 μg) in 10 mM Tris phosphate pH8.0, 120 mM NaCl, 0.1 mM EGTA, 5 mM MgCl₂, is iodinated by reaction with1 mCi carrier-free Na ¹²⁵I (NEN Life Science Products, Boston, Mass.) inthe presence of IODO-BEADS™ (Pierce, Rockford, Ill.) for 15 min at 4° C.The radiolabeled protein is separated from free Na¹²⁵I on a spin column(BioRad Bio-spin 6; Bio-Rad Laboratories, Hercules, Calif.) and storedin aliquots at −80° C. until use.

[0286] After incubation, binding reactions are filtered through a 5 μmfilter (hydrophilic Durapore PVDF filter; Millipore Corp., Bedford,Mass.) adapted to a multiwell format, to separate bound and unbound¹²⁵I-calcineurin. The filters are rapidly washed under continuous vacuumwith 50 mM HEPES pH 7.0, 150 mM NaCl, 5 mM MgCl₂, 200 μM CaCl₂, 10%glycerol, 1% Triton X-100. The ¹²⁵I-calcineurin retained by theimmobilized GST fusion proteins is quantified by scintillation countingusing a gamma counter. A candidate agent is scored as positive if itreduces the protein-protein interaction of calcineurin and NFAT asassessed by radiolabel bound in the assay.

[0287] Those skilled in the art will be aware of many alternative waysto carry out an equivalent assay. For example, the reactions can besuitably modified to use any of the proteins, protein fragments,peptides, or analogues described herein as materials for the assays. Theroles of the proteins can be reversed, so that calcineurin isimmobilized and NFAT is the radiolabeled compound. The reactions can becarried out with other solid supports or as a solution assay. Thoseskilled in the art will know that the optimal conditions of incubationand washing may change in such modified assays, and that smalladjustments of the conditions may be necessary, including, e.g., changesin the concentrations of proteins, the temperature, salts, pH, orinclusion of additional inhibitors of peptidases and phosphatases in theincubation buffer. Likewise, use of other resins or solid supports mayrequire the inclusion in the incubation of substances to blocknonspecific binding to these materials.

[0288] This method of screening is capable of identifying organicmolecule inhibitors, e.g., that bind to calcineurin and thereby inhibitthe protein-protein interaction of calcineurin and NFAT. Skilledpractitioners will also appreciate that this method, and other methodsdescribed herein (e.g., the method described in Example 8, below), canbe used to identify inhibitors that bind to NFAT, instead of or inaddition to calcineurin, and that such organic molecules can be used toinhibit the protein-protein interaction of calcineurin and NFAT.

Example 8 High-Throughput Screen for Inhibitors of Protein-ProteinInteraction Between Calcineurin and NFAT (Without a Washing Step)

[0289] This example describes a high-throughput screen for inhibitors ofprotein-protein interaction between calcineurin and NFAT using ascintillation proximity assay.

[0290] Recombinant NFAT1 is immobilized on SPA beads (obtained fromAmersham, Arlington Heights, Ill.)—e.g., by binding influenzahaemagglutinin-tagged NFAT1 to a mouse monoclonal antibody directedagainst the haemagglutinin epitope, and thereby to SPA beads derivatizedwith an anti-mouse antibody (Amersham, Arlington Heights, Ill.); or bybinding biotinylated NFAT1 to streptavidin SPA beads (Amersham)—and thebeads are washed in the binding buffer described in Example 7 anddistributed to replicate wells. Candidate agents are added as describedin Example 7, and the reactions for total binding and for inhibitionusing the 13-mer or 26-mer peptide are constituted as described inExample 7. Each reaction is supplemented with 100 μM CaCl₂, 800 nMcalmodulin, and 14 nM ¹²⁵I-calcineurin (final concentrations), and theincubation is carried out for 30 min at 4° C. Bound radioactivity isquantitated directly in the multiwell plate by scintillation counting. Acandidate agent is scored as positive if it reduces the protein-proteininteraction of calcineurin and NFAT as assessed by radiolabel bound inthe assay.

Example 9 High-Throughput Screen for Inhibitors of Dephosphorylation ofNFAT by Calcineurin

[0291] This example illustrates a high-throughput screen for inhibitorsof dephosphorylation of NFAT by calcineurin.

[0292] Hexahistidine-tagged human NFAT1(1-415) is purified frombacterial lysates by incubation with Ni²⁺-NTA-agarose in 50 mM Tris pH8.0, 150 mM NaCl, for 30 min at 4° C. For in vitro labeling with ³²P,NFAT bound on the agarose beads is incubated in 20 mM HEPES pH 7.5, 20mM MgCl₂, 20 μM unlabelled ATP with the addition of 0.17 mCi/mlγ-³²P-ATP (NEN Life Science Products, Boston, Mass.) and 1700 units/mlof the MAP kinase ERK2 (New England Biolabs, Beverly, Mass.). After 20min at 30° C., the beads are thoroughly washed to remove the kinase andunincorporated radiolabel, resuspended in phosphatase buffer (50 mMHEPES pH 7.5, 140 mM NaCl, 2 mM MnCl₂, 2 mM CaCl₂, 15 mM2-mercaptoethanol), and distributed to multiple wells. Candidate agents,or phosphatase buffer or diluent only, are added to individualreactions. The inhibitory 13-mer peptide or 26-mer peptide describedherein is added to individual reactions in a range of finalconcentrations (100 μM to 2 mM) to serve as positive controls forinhibition. Each reaction is brought to a final volume of 30 μl with theaddition of 150 nM calcineurin (Sigma Chemical Co., St. Louis, Mo.) and500 nM calmodulin (Sigma), and incubated 20 min at 30° C. Thesupernatant is collected by filtration into a multiwell plate, andreleased ³²P-phosphate is determined by scintillation counting.

[0293] Other formats for the assay involve different methods ofseparating free phosphate from phosphate covalently bound to protein,utilization of a variety of NFAT substrates, or utilization of NFATpurified from cell extracts. Likewise, radiolabel remaining bound toprotein may be measured rather than measuring the radiolabel released.In some embodiments, a chromogenic assay for free phosphate (EnzChekPhosphate Assay Kit; Molecular Probes, Eugene, Oreg.) may be substitutedfor the radioactive assay, avoiding the use of radioactivity and theneed for separation of free phosphate from protein after the incubationwith calcineurin.

[0294] Other protein kinases are also suitable for preparation of³²P-labelled NFAT if they incorporate phosphate at sites that aretargets for dephosphorylation by calcineurin, and if dephosphorylationof those sites by calcineurin is inhibited by the 13-mer or 26-merinhibitory peptides described herein as is characteristic of thephysiological dephosphorylation by calcineurin. In using other proteinkinases, the particular conditions of the labeling reaction will dependon the optimal conditions for enzymatic activity of the kinase used.Likewise, for an optimal assay with different preparations ofcalcineurin, the concentration of calcineurin or of buffer componentssuch as divalent ions, or reaction time or temperature, may requireadjustments that can be determined by routine experimentation.

Example 10 Detection of NFAT Dephosphorylation by Calcineurin UsingAntibodies to a Dephosphorylated NFAT Peptide

[0295] Phosphorylated NFAT1 or HA-tagged phosphorylated NFAT1 indephosphorylation buffer (80 μl 100 mM HEPES pH 7.4, 100 mM NaCl, 20 mMpotassium acetate, 2 mM magnesium acetate, 2 mM dithiothreitol, 0.1mg/ml bovine serum albumin) is distributed, 0.3 ng/well, to the wells ofa multiwell plate. To individual wells is added 20 μl ofdephosphorylation buffer alone, of buffer containing a compound to betested, or of buffer containing 13-mer or 26-mer inhibitory peptide(1-500 μM). Each reaction is brought to a final volume of 120 μl and afinal concentration of 1 mM CaCl₂, 150 nM calcineurin (Sigma ChemicalCo., St. Louis, Mo.) and 500 nM calmodulin (Sigma Chemical Co., St.Louis, Mo.), and incubated 20 min at 30° C. The reaction is stopped bythe addition of the calcineurin inhibitors EGTA and sodium pyrophosphateto concentrations of 5 mM and 30 mM, respectively. The contents of eachwell are transferred to a second multiwell plate coated for an ELISAwith anti-dephosphopeptide antibody, and dephosphorylated NFAT isallowed to bind for 3 h at 20° C. The wells are washed three times withphosphate-buffered saline pH 7; incubated with alkalinephosphatase-labeled anti-67.1 antibody (Ho et al., J Biol Chem269:28181-28186 (1994)) or antiHA tag antibody, as appropriate, 1 h at20° C.; and again washed three times with phosphate-buffered saline pH7. Reaction buffer containing the substrate p-nitrophenyl phosphate isadded, the alkaline phosphatase reaction is allowed to proceed at 20° C.until color develops in the control samples that were dephosphorylatedby calcineurin in the absence of an inhibitor, the reaction is stoppedwith 3 N NaOH, and absorbance is read at 405 nm. Compounds that inhibitthe calcineurin-NFAT interaction are detected by a decreased absorbance,with a threshold decrease for example of 30%.

Example 11 Detection of the Calcineurin-Dependent Change inIntracellular Localization of NFAT1 Using an Antibody

[0296] Cells expressing NFAT1, e.g. PC12 cells, are plated in L15CO₂medium (Nardone et al., Proc Natl Acad Sci USA 91:4412-4416 (1994)) intoreplicate wells coated with poly-D-lysine in a 96-well plate. Foroptimal visualization the cells are allowed to attach to the substrateovernight. Individual wells are preincubated 20 min at 37° C. withmedium alone, with medium containing a compound to be tested, or withmedium containing known inhibitors of NFAT activation; and then furtherincubated 20 min at 37° C. with stimulus, e.g. ionomycin 20 μM for PC12cells, in the continuing presence of test compound or inhibitor in thosewells where a test compound or inhibitor is used. The assay isterminated by removal of medium and addition of fixative, 4%paraformaldehyde in 0.12 M phosphate buffer, and fixation is allowed toproceed 30 min at room temperature. The wells are washed 4 times withphosphate-buffered saline, and the fixed cells are permeabilized andpreblocked for 30 min at room temperature with phosphate-buffered salinecontaining 5% fetal calf serum and 0.3% Triton X-100. The primaryantibody incubation for immune staining is with anti-67.1 antiserum (Hoet al., J Biol Chem 269:28181-28186 (1994)), followed by washing andincubation with Cy3™-labeled donkey anti-rabbit IgG (JacksonImmunoResearch Laboratories, West Grove, Pa.). After washing to removeunbound second antibody, the samples are examined by fluorescencemicroscopy to determine the localization of immune staining for NFAT1,and by phase contrast microscopy to visualize cell nuclei and cytoplasm.With adequate stimulation, as verified by examination of those samplesincubated with stimulus alone, not more than −1% of cells should havecytoplasmic NFAT1. A tested compound is scored positive if a largerfraction of cells displays predominantly cytoplasmic staining for NFAT1,or if significant numbers of cells display some cytoplasmic retention ofNFAT1.

Example 12 Detection of the Calcineurin-Dependent Change inIntracellular Localization of NFAT1 Using an NFAT-GFP Fusion Protein

[0297] HeLa cell line NFAT16, a cell line stably expressingGFP-NFAT1(1-460) under control of the CMV promoter, is plated in a96-well plate suitable for subsequent fluorescence microscopy, andincubated overnight in Dulbecco's modified Eagle medium supplementedwith 10% fetal calf serum, 10 mM HEPES, 2 mM L-glutamine, and 1 mg/mlneomycin to permit spreading of cells on the substrate for optimalvisualization. A preincubation is initiated by withdrawal of the growthmedium, and addition of the same medium containing (in individual wells)either a compound to be tested, a known inhibitor of NFAT activation andnuclear translocation (e.g., CsA), or no additive. After a 30 minpreincubation, the incubation is supplemented with an additional 3 mMCaCl₂ and with ionomycin to 3 μM final concentration, except that noionomycin is added to the designated unstimulated control wells. Thepurpose of the additional Ca²⁺ is to ensure optimal activation of NFATin this cell line. The incubation is continued at 37° C. for 10 min. Themedium is aspirated, and the cells are fixed by treatment with 3%paraformaldehyde in 0.1 M sodium phosphate, pH 7.4, for 30 min at roomtemperature. Fixative is removed by washing three times, 5 min each,with phosphate-buffered saline. Localization of NFAT1(1-460)-GFP in thecells is examined by fluorescence microscopy using suitable excitationand emission filters for GFP, and nuclei and cytoplasm are visualized byphase contrast microscopy. Less than 5% of stimulated NFAT16 cells showpredominantly cytoplasmic NFAT-GFP fluorescence under the statedconditions. A tested compound is scored positive if a larger fraction ofcells, for example more than 10% of cells, displays predominantlycytoplasmic staining for NFAT1.

[0298] Many available cell lines, primary cells, or cells expressingrecombinant NFAT display a calcineurin-dependent translocation of NFATto the cell nucleus. Those skilled in the art will know that foradequate stimulation of different cell types, adjustments are made inthe conditions of the assay, e.g., in the stimulus used, theconcentration of stimulus, the time of incubation with stimulus, and theaddition of CaCl₂. In each case, appropriate assay conditions for thecells studied can be determined by routine experimentation.

Example 13 Selection of a Consensus Peptide Sequence that isCharacteristic of Peptides that Bind Tightly to the SPRIEITPS-BindingSite of Calcineurin

[0299] This example illustrates the selection from combinatorial peptidelibraries of peptide analogues of the SPRIEITPS peptide that displayenhanced affinity for calcineurin.

[0300] The basic strategy was to select peptides from combinatorialpeptide libraries based on their ability to bind to a GST fusion proteincontaining the calcineurin catalytic domain. The peptide libraries werebiased toward binding at the NFAT recognition site on calcineurin byincorporating a partial consensus sequence. For example, as will beexplained in more detail below, the first peptide library incorporatedthe calcineurin-binding PxIxIT sequence motif that is conserved in theNFAT proteins (FIG. 4A). The combinatorial peptide library methoddepends on the fact that the presence of certain amino acid residues, atan individual position within the peptide that contacts calcineurin orthat influences the peptide conformation necessary for efficientinteraction with calcineurin, will confer enhanced binding to the targetsite, whereas the presence of certain other amino acid residues willimpair binding to the site.

[0301] In more detail, combinatorial peptide libraries were synthesizedat the Tufts-New England Medical Center Peptide Synthesis Facility usingN-α-fluorenyl methoxycarbonyl-protected amino acids and standardBOP/HOBt coupling chemistry as described (M B Yaffe et al (1997) Cell91, 961-971). GST-calcineurin is a glutathione S-transferase andcalcineurin catalytic domain fusion protein, which was expressed from aplasmid derived by cloning a DNA fragment encoding residues 2-347 ofhuman calcineurin Aα (GenBank accession number L14778) in-frame at theBamHI site of pGEX-6P vector (Amersham Pharmacia Biotech). The codingsequence of the abbreviated calcineurin cDNA was altered to encode aC-terminal peptide HPSWAPNFD, in place of HPYWLPNFM, in order to enhancestability of the truncated protein. The nucleotide and amino acidsequences of this modified calcineurin catalytic domain are shown inFIGS. 3A and 3B, respectively.

[0302] Library screening was performed using approximately 1 mg ofGST-calcineurin fusion protein immobilized on 100 μl ofglutathione-agarose resin in buffer containing 50 mM HEPES pH 7.5, 150mM NaCl, 5 mM MgCl₂, 300 μM CaCl₂, 100 μM sodium vanadate, 100 μM EGTA,1 mM dithiothreitol, and 1% Triton X-100. After allowing the mixture ofpeptides to bind to the immobilized protein, the resin was washed twicewith buffer and twice with PBS, bound peptides were eluted with 30%acetic acid, and the mixture of eluted peptides was sequenced. Analiquot of the peptide library that had not been selected on thecalcineurin affinity resin was also sequenced to provide baseline dataon the recovery of individual amino acids at each randomized position. Anumerical index of selection (“preference value”) for each amino acidresidue at a given randomized position was obtained (M B Yaffe et al(1997) Cell 91, 961-971; Yaffe M B and Cantley L C (2000) MethodsEnzymol 328, 157-170) by comparing the relative abundance (mole percent)of that residue at the corresponding sequencing cycle of the selectedpeptide mixture with the relative abundance at the same sequencing cycleof the unselected library. A preference value greater than 1.0 indicatesselection on the calcineurin column, and a preference value of around2.0 or greater indicates moderately strong to strong selection (M BYaffe et al (1997) Cell 91, 961-971; Yaffe M B and Cantley L C (2000)Methods Enzymol 328, 157-170).

[0303] FIGS. 4A-4D depict a summary of the evolution of an optimizedpeptide inhibitor capable of disrupting the NFAT-calcineurininteraction. A first combinatorial peptide library, with the sequenceMAxxxPxIxITxxHKK, was targeted to the docking site on calcineurin byincorporation of the sequence PxIxIT (see FIG. 4A) and was randomized atseven positions where the sequence is not fully conserved within theNFAT family (FIG. 4B). In FIG. 4B, residues in the nondegeneratepositions of the peptide library are shown in the single-letter code,and the essential targeting residues are boxed. Within the library, inaggregate, the randomized positions denoted “X” contained roughlyequimolar amounts of all naturally-occurring amino acid residues exceptcysteine. This first library was selected for binding toGST-calcineurin, and the data were analyzed as previously described.Amino acid residues selected at the degenerate positions of the firstlibrary are shown in FIG. 4B, with residues that were strongly selectedpresented in bold face type and underlined, and with preference valuesindicated in parentheses. Thus, in the first library, the peptide pooleluted from the GST-calcineurin column showed moderate selection forglycine, serine, and lysine at position 3; no preferred residues atposition 4; selection for histidine or aliphatic residues at position 5;and moderate selection for the polar residues threonine, lysine,glutamine, and glutamate at position 7. At position 9 there wasrelatively weak selection for aliphatic residues, notably valine. Atpositions 12 and 13 there was selection for glycine and prolinerespectively, suggesting that the NFAT binding site in calcineurinimposes a turn at the C-terminal end of the PxIxIT motif.

[0304] To refine the peptide selection further, a second degeneratepeptide library was synthesized, in this case targeted to the samebinding site on calcineurin by incorporation of an alternative set offixed residues based on the initial round of library selection, but nowrandomized at positions that had been fixed previously (FIG. 4C). InFIG. 4C, the new targeting residues are boxed, and the randomizedpositions are again indicated by “X”. “Z” denotes a mixture of thenonnatural amino acid residues p-fluorophenylalanine,p-chlorophenylalanine, 2-naphthylalanine,tetrahydroisoquinoline-3-carboxylic acid, and cyclohexylalanine. As isevident in this figure, positions 3 and 5 were fixed as glycine andhistidine, respectively, while positions 12 and 13 were fixed as glycineand proline to favor the putative turn. Position 7 contained 50%threonine and 50% a mixture of all other residues, to probe whetherthreonine and polar residues were indeed preferred at this position.Position 8 contained the 19 natural amino acid residues (omittingcysteine) and 5 additional nonnatural amino acid residues with largearomatic or cyclic groups, to determine whether binding affinity couldbe improved by substitution of a large hydrophobic side chain for theisoleucine side chain that is naturally conserved at this position.Position 10 was fixed as isoleucine, positions 9 and 11 were randomized,and the two C-terminal lysine residues of the first library werereplaced by glutamate residues to eliminate any bias that might havebeen present in the first library because of their positive charge.

[0305] Selection of the second library over the calcineurin affinitymatrix yielded strongly preferred residues at most of the randomizedpositions (FIG. 4C). A striking feature in this round of selection wasthat bulky or β-branched hydrophobic residues (valine, isoleucine,leucine) were highly preferred at positions 7 and 9 for binding to thetarget site. Proline was preferred at position 4, echoing its occurrenceat this position in NFAT1, although not in NFAT2-NFAT4. Isoleucine wasstringently preferred at position 8, with lesser selection for otherhydrophobic residues and no selection for the nonnatural amino acidresidues, consistent with the invariance of isoleucine at this positionof the PxIxIT sequence in all four NFAT proteins. Finally, there wasstrong selection for the conserved threonine of the PxIxIT sequence atposition 11, with a weaker preference for serine. The findings can besummarized in the sequence of a consensus peptide, MAGPHPVIVITGPHEE(FIG. 4D).

[0306] These results identified a consensus peptide sequence, containingthe PxIxIT motif, that is characteristic of peptides that bind tightlyto the SPRIEITPS-binding site of calcineurin.

Example 14 VIVIT Peptide Strongly Inhibits Dephosphorylation of NFAT,but does not Inhibit Calcineurin Enzyme Activity

[0307] This example illustrates that a synthetic peptide with theconsensus sequence determined in Example 13, MAGPHPVIVITGPHEE, is aneffective inhibitor of NFAT recognition and dephosphorylation, and likeSPRIEITPS does not interfere with calcineurin enzymatic activitygenerally.

[0308] Based on the results described in Example 13, a predicted optimalpeptide was synthesized, with the amino acid sequence MAGPHPVIVITGPHEE,and its effect on the interaction of calcineurin with NFAT was examinedin biochemical assays. This peptide is referred to hereinafter as “VIVIT16-mer peptide”, and this peptide and other peptides based on theconsensus or alternative consensus residues determined in Example 13 aregenerically referred to as “VIVIT” peptides.

[0309] FIGS. 5A-5C illustrate that the optimised VIVIT peptide is apotent inhibitor of the interaction between NFAT and calcineurin. InFIG. 5A, the protein-protein interaction between calcineurin and NFATwas assayed by incubating 200 nM calcineurin that had been activatedwith 600 nM calmodulin and 2 mM CaCl₂ with 3 μg GST or GST-NFAT1(1-415)as described (F J Garcia-Cozar et al (1998) J Biol Chem 273,23877-23883). NFAT1-bound calcineurin was collected by incubation withglutathione-Sepharose resin and detected by Western blotting with apolyclonal antibody to the calcineurin A chain (C Loh et al (1996) JBiol Chem 271, 10884-10891). The peptides SPRIEIT-13 or SPAIAIA-25 (JAramburu et al (1998) Mol Cell 1, 627-637) or the VIVIT 16-mer peptidewere included in the assay where indicated, in the amounts indicatedabove each lane (in μM).

[0310] The assay demonstrated specific calcineurin binding to GST-NFAT1,but not to GST (FIG. 5A, compare lane 2 with lane 1, respectively). Boththe SPRIEITPS peptide (lanes 3-6), which recapitulates the dockingsequence in NFAT1, and the VIVIT 16-mer peptide (lanes 7-10) inhibitedthe binding of activated calcineurin to GST-NFAT1. The mutant peptideSPAIAIA did not inhibit calcineurin binding to GST-NFAT1 when tested at100 μM (compare lane 11 with lane 2).

[0311] The VIVIT 16-mer peptide also inhibited the calcineurin-mediateddephosphorylation of NFAT (FIG. 5B). Lysates of HeLa cells stablyexpressing HA-NFAT1(1-460)-GFP (hereinafter “HA-NFAT1”) were incubatedwith 200 nM calcineurin plus 600 nM calmodulin and 2 mM CaCl₂ for 30 minat 30 C. in buffer as described (J Aramburu et al (1998) Mol Cell 1,627-637). The phosphorylation status of NFAT was evaluated bySDS-polyacrylamide gel electrophoresis and Western blotting with anti-HAantibody 12CA5. For these experiments, reaction conditions were chosenthat produced only limited dephosphorylation of NFAT (FIG. 5B, lane 2)in order to maximize the sensitivity of the assay in detectinginhibition. The SPRIEIT-13 (lanes 3-6), VIVIT 16-mer (lanes 7-9), andSPAIAIA-25 (lane 10) peptides were again included in the assay whereindicated, in the amounts indicated above each lane (in μM). Todemonstrate the effect of completely inhibiting calcineurin phosphataseactivity, the general phosphatase inhibitor sodium pyrophosphate (NaPPI)was used at 10 mM (lane 1). Both the VIVIT peptide and the SPRIEITPSpeptide were effective at inhibiting NFAT dephosphorylation.

[0312] Inhibition of NFAT dephosphorylation by VIVIT peptide was not dueto occlusion of the catalytic site of calcineurin. This point hadpreviously been established for the SPRIEITPS peptide (see Example 4),and was tested here by measuring calcineurin phosphatase activity with³²P-labelled phospho-RII peptide as substrate (J Aramburu et al (1998)Mol Cell 1, 627-637). SPRIEIT-13, SPAIAIA-25, and VIVIT 16-mer peptideswere included in the incubation as indicated. The results are plotted inFIG. 5C as radiolabel released from ³²P-phospho-RII peptide (counts perminute×10⁻³). Where peptide was present, its concentration (μM) isindicated in FIG. 5C; CsA/cyclophilin A complexes (CsA/Cyp) were presentat 10 μM. Even at 100 μM, a concentration that completely blockscalcineurin-NFAT1 binding and NFAT1 dephosphorylation (FIGS. 5A and 5B),the VIVIT peptide did not inhibit calcineurin phosphatase activitytowards the RII phosphopeptide (FIG. 5C). In contrast, CsA/cyclophilin Acomplexes inhibited calcineurin phosphatase activity by approximately95% in this assay, consistent with the established mode of action ofCsA/cyclophilin A complexes, which is interference with calcineurinactivity against all its peptide and protein substrates. These resultsshowed that VIVIT peptide is a selective inhibitor, and blocks thedephosphorylation of NFAT but not of RII phosphopeptide.

Example 15 VIVIT Peptide Selectively Inhibits NFAT-Dependent Expressionof Reporter Genes

[0313] This example illustrates that expression of a fusion proteinincluding the VIVIT 16-mer peptide selectively inhibits NFAT-mediatedreporter gene expression, but not NF-κB mediated reporter geneexpression, in T lymphocytes.

[0314] The general strategy in this Example was to examinetranscriptional signalling in cells having effective concentrations ofVIVIT peptide in the cell cytoplasm. Since the VIVIT peptide itself isnot cell permeant, it was delivered into cells by expression of aGFP-VIVIT fusion protein. Luciferase reporter plasmids werecotransfected into the same cells, and the effects of VIVIT peptide onNFAT-dependent and NF-κB-dependent transcription were examined afterappropriate stimulation of the cells.

[0315] In more detail, Jurkat cells were transfected (J Aramburu et al(1998) Mol Cell 1, 627-637; C Luo et al (1996) Mol Cell Biol 16,3955-3966) with 0.25 μg per 10⁶ cells luciferase reporter plasmid; with0.25 μg per 10⁶ cells expression plasmid for NFAT1, NFAT2, or NFAT4;with 40 ng per 10⁶ cells human growth hormone (hGH) expression plasmidas an internal reference for transfection efficiency; and with 0.5 μgper 10 cells, or other indicated amount, GFP, GFP-SPRIEIT, or GFP-VIVITexpression plasmids. The reporter plasmids contained three tandem copiesof the distal NFAT-AP 1 site of the murine IL-2 promoter (3×NFAT-Luc),two copies of a consensus NF-κB site (2×NF-κB-Luc), the human IL-2promoter, or the human TNFα promoter (C Luo et al (1996) Mol Cell Biol16, 3955-3966; F Mercurio et al (1997) Science 278, 860-866). The GFPexpression vector was pEGFP-N1 (Clontech). The GFP-SPRIEIT expressionvector was previously described (J Aramburu et al (1998) Mol Cell 1,627-637), and the GFP-VIVIT expression vector was constructed bysubcloning an oligonucleotide encoding the sequence MAGPHPVIVITGPHEEin-frame with green fluorescent protein (GFP) at the N-terminus of GFP.Twenty-four hours after transfection, cells were left unstimulated,stimulated for 6 hours with 20 nM phorbol 12-myristate 13-acetate (PMA)and 1 μM ionomycin, or stimulated for 6 hours with 0.2 μg/ml immobilizedanti-CD3 (HIT3a, Pharmingen) and 0.5 μg/ml soluble anti-CD28 (CD28.2,Pharmingen). CsA was added 30 min before the stimuli. Luciferaseactivity in cell lysates was measured using a luciferase assay system(Promega) and was normalized to the concentration of hGH in the cellculture medium determined with a radioimmunometric assay kit(Hybritech).

[0316] FIGS. 6A-6D are bar graphs that generally illustrate that VIVITpeptide is a selective and potent inhibitor of NFAT activity, and thatit does not substantially affect calcineurin activity or othercalcineurin-dependent processes. FIG. 6A illustrates that VIVIT peptideinhibits NFAT-dependent gene expression. In the left panel panel of FIG.6A are graphed data from Jurkat cells that had been cotransfected with a3×NFAT-Luc reporter plasmid and with expression plasmids encoding murineNFAT1, GFP, GFP-SPRIEIT, or GFP-VIVIT as indicated. In the right panelare data from Jurkat cells that had been cotransfected with a 3×NFAT-Lucreporter plasmid and with expression plasmids encoding murine NFAT1,human NFAT2, or human NFAT4 as indicated. Twenty-four hours aftertransfection, cells were left unstimulated (Unstimulated; open bars) orwere stimulated for 6 hours with PMA and ionomycin (PMA+iono; solidbars), cell lysates were prepared, and luciferase activity attributableto endogenous NFAT (Endog.) or to overexpressed NFAT proteins wasmeasured.

[0317] Little luciferase was detected in lysates from unstimulatedcells. As shown in the left panel of FIG. 6A, stimulation with PMA andionomycin induced luciferase through activation of endogenous NFAT, andthe induced expression was partially blocked by SPRIEIT and almostcompletely blocked by VIVIT. Even when NFAT1, NFAT2, or NFAT4 wasoverexpressed in the cells (right panel of FIG. 6A), VIVIT efficientlyinhibited luciferase expression. Since luciferase induction is largelyattributable to an individual NFAT protein in the latter cases, thistest provides clear confirmation that the cellular action of VIVIT is onthe calcineurin-NFAT pathway.

[0318]FIG. 6B illustrates that delivery of increasing concentrations ofthe GFP-VIVIT expression plasmid inhibited activation of an NFATreporter but not activation of an NF-KB reporter. Jurkat cells werecotransfected with 3×NFAT-Luc (left panel) or with 2×NF-κB-Luc (rightpanel) reporter plasmid, and with the indicated amounts (μg plasmid per10⁶ cells) of GFP and GFP-VIVIT expression plasmids, the total amount ofplasmid DNA added being kept constant. Twenty-four hours aftertransfection, cells were left untreated (open bars) or were stimulatedfor 6 hours with PMA and ionomycin (solid bars).

[0319] These data show that basal levels of luciferase were unaffectedby VIVIT peptide, but that stimulated expression of the NFAT reportergene was blocked by VIVIT. The maximal effect of GFP-VIVIT onNFAT-dependent reporter activity was as great as that of CsA, inhibitingreporter activity to the level observed in unstimulated cells. On theother hand, stimulated expression of NF-κB reporter was unaffected byVIVIT.

[0320] The finding that activation of an NF-κB reporter is not affectedby VIVIT is of special interest, because NF-κB activation in T cells iscalcineurin-dependent and is sensitive to FK506 and CsA (B Frantz et al(1994) EMBO J 13, 861-870; K Kalli et al (1998) Mol Cell Biol 18,3140-3148). The calcineurin dependence of reporter activity was verifiedin Jurkat T cells transfected with 3×NFAT-Luc and 2×NF-κB-Luc reporterplasmids (FIG. 6C, left and right panels, respectively). Cells wereuntreated, or were stimulated with PMA and ionomycin (P+I), in theabsence or presence of CsA at the concentrations indicated. CsA in factproduced a concentration-dependent inhibition of NF-κB reporter geneexpression, with IC₅₀ less than 5 nM and maximal inhibition at 100 nM.This set of experiments indicates that calcineurin-NFKB signalling isfully functional in cells expressing VIVIT, and that VIVIT has a moreselective action than CsA, discriminating between transcriptionalsignalling pathways downstream of calcineurin.

[0321]FIG. 6D illustrates that VIVIT peptide inhibited NFAT-dependentactivation of the IL-2 and TNFα promoters (FIG. 6D). Jurkat cells werecotransfected with GFP or GFP-VIVIT expression plasmid and with aluciferase reporter plasmid driven either by the human IL-2 promoter(left panel) or by the human TNFα promoter (right panel). Cells wereunstimulated (open bars), stimulated with PMA and ionomycin (solidbars), or stimulated with anti-CD3 and anti-CD28 (hatched bars).GFP-VIVIT, but not GFP, efficiently inhibited gene expression driven bythe IL-2 and TNFα promoters in T cells stimulated with PMA and ionomycinor with anti-CD3 plus anti-CD28. These results showed that VIVIT peptideinhibits calcineurin-NFAT transcriptional signalling in T cells, andmoreover that VIVIT peptide is a more selective inhibitor than CsA,since it does not inhibit calcineurin-NF-κB transcriptional signallingin T cells.

Example 16 VIVIT Peptide Inhibits Expression of NFAT-Dependent, but notNFAT-Independent, Cyclosporin-Sensitive Genes

[0322] This example illustrates that expression of a fusion proteinincluding the VIVIT 16-mer peptide selectively inhibits NFAT-mediatedexpression of endogenous cytokine genes in T lymphocytes.

[0323] Studying the effect of VIVIT peptide on endogenous geneexpression presents the challenge that the cell population examined mustuniformly express VIVIT. Thus, to determine whether the VIVIT peptideinhibits expression of endogenous NFAT-dependent genes, Jurkat T cellswere cotransfected with GFP or GFP-VIVIT expression plasmids, togetherwith an expression plasmid encoding a selectable cell surface marker(murine CD4) that allowed isolation of a homogeneous population oftransfected cells. The purified cells were then stimulated, and theinduced expression of multiple cytokine mRNAs was analyzed by multiprobeRNase protection assay.

[0324] In a more detail, human Jurkat T cells were cotransfected with0.75 μg per 10⁶ cells murine CD4 expression plasmid (mCD4/mL3T4, kindgift of Dr Dan R Littman, New York University) and 0.75 μg per 10⁶ cellsGFP or GFP-VIVIT expression plasmid. Cells expressing mCD4 were selectedusing magnetic beads (Dynabeads L3T4, Dynal Biotech) as described (AKiani et al (1997) Immunity 7, 849-860). Magnetic bead selection ofmCD4-expressing cells yielded a population that was greater than 90%GFP-positive, whereas mCD4-nonexpressing cells were less than 5%GFP-positive, indicating that the selection isolated the desiredpopulation of cells expressing GFP or GFP-VIVIT. RNA was prepared fromthe selected cells, and multiprobe RNase protection assays wereperformed using RiboQuant multiprobe kits (Pharmingen, San Diego,Calif.) as described (A Kiani et al (1997) Immunity 7, 849-860). Theresults are shown in FIGS. 7A to 7C.

[0325] FIGS. 7A-7C are pictures of autoradiograms of RNA gels generallyillustrating that VIVIT peptide blocks NFAT-dependent expression ofendogenous cytokine genes. FIGS. 7A-7C show protected segments ofcomplementary RNA probes, which indicate the presence and level ofspecific cytokine mRNAs in the corresponding samples. In FIG. 7A, lanes1-4 represent Jurkat T cells that had not been transfected withexpression plasmids. For example, FIG. 7A shows that IL-2 and IL-13mRNAs were undetectable in resting cells (lane 1) and were induced incells stimulated with PMA and ionomycin (lane 2). The mRNAs wereundetectable in resting cells treated with CsA (lane 3), and theirinduction was prevented in the presence of CsA (lane 4). Cellstransfected with mCD4 expression vector only, and selected usingmagnetic beads, did not behave differently than the untransfected cellswith regard to expression of these cytokines (FIG. 7A, compare lanes 9and 10 with lanes 1 and 2). Thus, the presence of the cell surfaceprotein mCD4 and its interaction with anti-mCD4 on the magnetic beadsdid not in itself have a stimulatory or inhibitory influence on cytokinegene expression. Similarly, cells expressing unmodified GFP protein(together with mCD4) did not behave differently than cells without GFP(FIG. 7A, compare lanes 5 and 6 with lanes 9 and 10), or than Jurkatcells expressing no exogenous protein (FIG. 7A, compare lanes 5 and 6with lanes 1 and 2). In contrast, cells expressing GFP-VIVIT displayedmuch-attenuated induction of IL-2 and IL-13 mRNAs (FIG. 7A, comparelanes 7 and 8 with lanes 5 and 6). Levels of mRNA from the housekeepinggenes L32 and GAPDH, included as a control for sample loading, showedlittle variation from lane to lane.

[0326]FIG. 7B illustrates a similar analysis for the mRNAs encoding IL-3(which was also probed in the experiment of FIG. 7A), TNFα, GM-CSF, andMIP-1α. The untransfected cell controls are not shown for thisexperiment. The cytokine mRNAs probed were not detected in unstimulatedcells expressing mCD4 and selected with magnetic beads, but were inducedby treatment with PMA and ionomycin (FIG. 7B, lanes 5 and 6). Expressionof unmodified GFP had no effect on induction of the mRNAs (lanes 1 and2), but expression of GFP-VIVIT was strongly inhibitory (lanes 3 and 4).

[0327] In FIG. 7C, the first set of control cells had been transfectedwith the empty expression vectors (FIG. 7C, lanes 1-4). The mRNAsexamined, TNFβ, LT-β, and TNFα mRNAs, were induced by treatment with PMAand ionomycin (lanes 1 and 2), and their induction was largely orcompletely blocked by CsA (lanes 3 and 4). Basal and induced mRNA levelswere unaffected by GFP (lanes 5 and 6). TNFα mRNA induction was blockedby GFP-VIVIT, as it was in the experiment of FIG. 7B, but TNFβ andlymphotoxin-β (LT-β) mRNAs were insensitive to GFP-VIVIT (lanes 7 and8).

[0328] Thus the VIVIT peptide inhibited inducible expression of IL-2,IL-13, IL-3, TNFα, GM-CSF, and MIP-1α mRNAs in Jurkat T cells,consistent with published work indicating the presence of functionalNFAT sites in the promoter/enhancer regions of these genes (see A Rao etal (1997) Annu Rev Immunol 15, 707-747; G R Crabtree (1999) Cell 96,611-614). In contrast, the expression of TNFβ and lymphotoxin-β,although sensitive to cyclosporin A, was unaffected by VIVIT peptide.This latter result, demonstrating signalling that is inhibited by CsAand not inhibited by VIVIT, is most easily understood as reflectingcalcineurin transcriptional signalling pathway(s) that are not routedthrough NFAT.

[0329] These results show that VIVIT peptide inhibits transcriptionalsignalling through the calcineurin-NFAT pathway, but not through othercalcineurin-dependent pathways, in T cells.

Example 17 High-Throughput Screening Assay for Inhibitors of theProtein-Peptide Interaction Between Calcineurin and VIVIT Peptide

[0330] This example illustrates the validation of a high-throughputscreening assay for compounds that inhibit binding of the optimizedVIVIT peptide to calcineurin.

[0331] The principle of the assay is to assess binding to calcineurin ofa VIVIT peptide, covalently labelled with the fluorophore Oregon Green™488, by measuring the polarization of Oregon Green fluorescence. Thefluorescence lifetime of Oregon Green is ˜4 ns, and this label istherefore well suited (see, for example, C R Cantor and P R Schimmel(1980) Biophysical Chemistry, Part II: Techniques for the study ofbiological structure and function, pp. 454-465, W. H. Freeman andCompany, San Francisco, 1980) to discriminate between rotationalmovements of the free VIVIT peptide and the peptide bound to arecombinant calcineurin with MW ˜40 kDa.

[0332] The reagents utilized for the assay were a recombinant humancalcineurin catalytic domain and an Oregon Green-labelled VIVIT peptide.Recombinant calcineurin was expressed in E. coli strain DH5α as aglutathione S-transferase-calcineurin fusion protein, using anexpression construct prepared by subcloning the cDNA encoding a modifiedhuman calcineurin(2-347) (see FIGS. 3A-3B) into pGEX-6P. The calcineurincDNA coding sequence was altered to produce three amino substitutions(Y341 S, L343A, and M347D) near the carboxy terminus of the encodedprotein in order to enhance the stability of the truncated protein. TheGST-calcineurin fusion protein was purified from bacterial lysates bybatch affinity chromatography on glutathione-Sepharose resin, eluted,and dialyzed to remove low molecular weight contaminants.Calcineurin(2-347) was cleaved from the fusion protein usingPreScission™ protease (Amersham Pharmacia Biotech) under conditionsspecified by the supplier, and repurified on glutathione-Sepharose resinto remove GST and PreScission protease. The resulting human calcineurincatalytic domain had the sequence, not including an initial fourresidues encoded by the pGEX linker, shown in FIG. 3B. Unlabelled VIVIT14mer peptide (GPHPVIVITGPHEE-amide) was synthesized at Tufts-NewEngland Medical Center Peptide Synthesis Facility, purified by reversedphase HPLC, and the identity of the purified peptide verified by massspectrometry. VIVIT peptide (2 mg) was combined with 1.5 mg OregonGreen™ 488 carboxylic acid, succinimidyl ester 5-isomer (MolecularProbes) and 51 μl N,N-diisopropylethylamine (Aldrich) in 190 μlanhydrous N,N-dimethylformamide (Aldrich), and incubated at roomtemperature for 18 h in the dark with occasional mixing. The labelledpeptide, denoted OG-VIVIT, was purified by C18 reversed phase HPLC andits identity verified by mass spectrometry.

[0333] Saturable binding of OG-VIVIT to calcineurin was demonstrated byaddition of increasing concentrations of calcineurin to an assaysolution consisting of 30 nM OG-VIVIT in phosphate-buffered saline (PBS)containing 0.1% bovine gamma globulin (BGG) (Sigma). Triplicate samplesfor each concentration of calcineurin were transferred, 10 μl per well,to the wells of a black 384-well plate (LJL) and fluorescencepolarization determined employing an Analyst plate reader (MolecularDevices) using the fluorescein filter set. Results are expressed inmillipolarization units (mP), where polarization (P) is given, in termsof measured fluorescence intensities polarized parallel (I_(parallel))and perpendicular (I_(perpendicular)) to the exciting light, by thestandard definition:

P=(I_(parallel) − _(perpendicular))/(_(parallel) +I _(perpendicular)).

[0334] The typical polarization in this assay was 35-45 mP for freepeptide, and ˜200 mP for peptide bound to calcineurin. The data of FIG.8A indicate a saturable protein-peptide interaction with K_(d) ˜0.5 μM.

[0335] The ability of an unlabelled competitor to displace OG-VIVIT fromcalcineurin was demonstrated by addition of increasing concentrations ofunlabelled VIVIT peptide in an assay mixture consisting of 30 nMOG-VIVIT and 0.5 μM or 1 μM calcineurin(2-347) in PBS/0.1% BGG. Sampleswere analyzed as described in the preceding paragraph, and the resultingpolarization data were plotted against the concentration of unlabelledVIVIT peptide, as shown in FIG. 8B. Because a robust signal foruncompeted binding in this assay requires the use of calcineurin at aconcentration that is near or above its K_(d) for binding fluorescentVIVIT, the IC₅₀ measured in the assay is shifted slightly to higherconcentrations than the true K_(i). After correcting for this effect,the data were consistent with a K_(i) for unlabelled peptide binding of0.5 μM. The agreement between the K_(d) value measured for calcineurinin the direct binding assay and the K_(i) value measured for unlabelledpeptide in the competition assay is expected for protein-ligand bindingat a single, saturable site.

[0336] The binding assay was reproducible and showed little variationamong replicate wells. Assay quality was assessed as signal-to-noiseratio (S:N) and Z′ parameter using the expressions

S:N=(μ_(b)−μ_(f))/(σ_(b) ²+σ_(f) ²)^(0.5)

[0337] and

Z′=1−3 (σ_(b)+σ_(f))/(μ_(b)−μ_(f)),

[0338] respectively (J H Zhang et al (1999) J Biomol Screen 4, 67-73),where μ_(b) and μ_(f) are the mean polarization measured for bound andfree peptide, and σ_(b) and σ_(f) are the corresponding standarddeviations. Under the stated conditions, the assay had excellentcharacteristics, as expressed in a signal-to-noise ratio of 18.11 and aZ′ parameter of 0.78.

[0339] These results demonstrate that binding of a fluorescent VIVITpeptide to calcineurin can be detected by measurement of the change inits fluorescence polarization, and that competition by an unlabelledcompound included in the assay can be readily detected in a multiwellformat suitable for high-throughput screening.

Example 18 Identification of Inhibitors of the Calcineurin-VIVIT PeptideInteraction by High-Throughput Screening of a Diverse Chemical Library

[0340] This example illustrates the implementation of thehigh-throughput screening assay described in the preceding example toidentify low-molecular-weight organic molecules that inhibit thecalcineurin-VIVIT peptide interaction.

[0341] The fluorescence polarization assay for inhibitors ofcalcineurin-VIVIT interaction was used to screen a diverse chemicallibrary of 16320 compounds (DiverSet E library from ChemBridge). Theassay mixture, consisting of 30 nM OG-VIVIT and 0.5 μMcalcineurin(2-347) in PBS/0.1% BGG, was distributed in 10 μl aliquots tothe wells of black 384-well assay plates (LJL). In the case of freepeptide control samples, calcineurin was omitted. The library stockswere dissolved at a nominal concentration of 10 mM in DMSO, andformatted for convenient robotic transfer on 384-well plates at theInstitute of Chemistry and Cell Biology, Harvard Medical School. Toinitiate the competition assay, 40 nl volumes were pin-transferred fromeach well of a library stock plate to the corresponding wells of anassay plate, utilizing a robotic device. After a minimum 10 minincubation at room temperature to allow the binding to reachequilibrium, fluorescence polarization data were collected employing anAnalyst plate reader (Molecular Devices).

[0342] As a first step in data analysis, library compounds thatthemselves exhibited substantial fluorescence in the assay were excludedfrom consideration, since the polarization value obtained from thesesamples cannot accurately reflect the signal from OG-VIVIT. Librarycompounds that contributed substantially to the fluorescence signal wereidentified by plotting a histogram of total sample fluorescence, whichis proportional to (I_(parallel)+2I_(perpendicular)), where I_(parallel)and I_(perpendicular) are the fluorescence intensities parallel andperpendicular to the exciting light, and excluding those compounds whosetotal sample fluorescence exceeded the 99th percentile of a Gaussian fitto the main peak of OG-VIVIT fluorescence in the histogram of allsamples. A total of 13445 of the library compounds met this criterionand were further analyzed.

[0343] The signature of active compounds, i.e., those that displaceVIVIT peptide from calcineurin, is a decrease in polarization value, andhence the presence of the corresponding data points in the leftmost tailof the distribution of polarization values from the assay. Those librarycompounds that had not been excluded by the criterion of total samplefluorescence were ranked on the basis of their raw polarization scoresin the assay; on the basis of a percentile score for each samplerelative to a Gaussian fitted to a histogram of all samples on the sameplate; and on the basis of the projected IC₅₀ of the tested compoundcomputed from its nominal concentration of 40 μM, the knownconcentrations of calcineurin and fluorescent VIVIT peptide, and theknown K_(d) of the calcineurin-peptide interaction. For example, 66compounds (0.4% of the compounds screened) had a projected IC₅₀ below100 μM, which is a hit rate comparable to that in other high-throughputscreens. A set of 11 library compounds that ranked as best candidateinhibitors in the three ranking methods was selected for more detailedstudy.

[0344] Stocks of candidate inhibitor compounds and their structuralanalogues were purchased from ChemBridge or Asinex. The structure andpurity of individual compounds were verified by ¹H NMR, massspectrometry, or thin-layer chromatography. The selected compounds wereindividually tested at a series of concentrations in the fluorescencepolarization assay in which the compounds were incubated with 1 μMcalcineurin and 60 nM OG-VIVIT. Each of the 11 compounds produced aconcentration-dependent displacement of OG-VIVIT from calcineurin,confirming the results of the high-throughput screen. Illustrativecompetition data are shown in FIGS. 9A and 9B. The concentrations oftest compounds denoted INCA1 and INCA2 in FIG. 9A, and INCA6 in FIG. 9B,are plotted on the horizontal axis, and the polarization signal (mP)from OG-VIVIT is plotted on the vertical axis. Compound NEG2 in FIG. 9Bis one of several negative control compounds chosen from the chemicallibrary on the basis of their failure to inhibit calcineurin-peptideinteraction in the high-throughput screen. Displacement of OG-VIVIT fromcalcineurin was complete at high concentrations in the case of INCA1,INCA2, and INCA6, as shown in FIGS. 9A and 9B. With some othercompetitors the displacement of peptide was incomplete, reaching aplateau at 50-90% displacement. The latter compounds may beintrinsically less effective in displacing the peptide ligand, or may beinsoluble in aqueous buffer at the concentrations that would benecessary for full displacement. Because calcineurin concentration inthese experiments was not negligible relative to the K_(d) for OG-VIVITbinding and the probable K_(i) for some of the inhibitory compounds, thecompetition experiment was repeated for several inhibitory compounds andtheir structural analogues with varied calcineurin concentrations, andthe data fitted to a three-state equilibrium binding model (in whichbinding of VIVIT and inhibitor to calcineurin is mutually exclusive) ora four-state model (in which a ternary calcineurin-VIVIT-inhibitorcomplex is permitted) in order to estimate the K_(i) for binding of eachcompound to calcineurin. The precise K_(i) values estimated will dependon the model used, but the estimates vary within a relatively smallrange. For example, the K_(i) values estimated for INCA1 fell between0.5 μM and 2 μM. Compounds whose inhibitory activity was confirmed inthe secondary assay were termed “INCA” (inhibitor of NFAT-calcineurinassociation) coupled with a unique numerical designation for eachcompound. INCA1, INCA2, and INCA6 can be represented generally asfollows:

[0345] INCA 1:

[0346] wherein R₁ is H, R₂ is Ph, R₃ is C1, R₄ is CHAc₂, and R₅ is O;

[0347] INCA 2:

[0348] wherein R₁ is C₁, R₂ is C₁, R₃ is 0, R₄ is H, and R₅ is H; and

[0349] INCA 6:

[0350] wherein R₁ is O, R₂ is 4, R₃ is H, R₄ is 0, and R₅ is H.

[0351] In addition to INCA1, INCA2, and INCA6, several other compoundswere isolated using the screen described above, the structures of whichare presented in Appendix I. The estimated IC₅₀ and K_(i) values forINCA1, INCA2, and INCA6, as well the other isolated compounds, areprovided in Table 5, below. TABLE 5 Description of Calcineurin-NFATinhibitors Compound Estimated IC₅₀ (μM) K_(i) (μM) INCA1 1 0.5-2   INCA23 0.1-0.5  3 5 ˜20  4 10 Not determined  5 17 ˜20 INCA6 17 0.8-5    7 22 ˜5  8 25 ˜20  9 33 ˜100  10 38 ˜20 11 39 Not determined 12 43 ˜30 13 46Not determined 14 46 Not determined 15 48 Not determined 16 48 Notdetermined 17 49 Not determined 18 51 Not determined 19 51 ˜200  21 57Not determined 22 58 Not determined 30 72 Not determined 33 74 Notdetermined

[0352] These results demonstrate the isolation of a few compounds in adiverse chemical library that were able to inhibit the calcineurin-VIVITpeptide interaction when present at low micromolar concentrations, whichcan be further tested in established assays of calcineurin-NFATinteraction and NFAT activation.

Example 19 The inhibitors INCA1, INCA2, and INCA6 Bind to Calcineurin

[0353] This example illustrates that binding of several of the INCAcompounds to calcineurin detected in the absence of VIVIT peptideutilizing NMR spectroscopy.

[0354] In the fluorescence polarization assay, binding of the inhibitorycompounds to calcineurin is inferred from their ability to displacefluorescent VIVIT peptide from the protein. To demonstrate independentlythat these compounds bind to calcineurin, NMR titration spectroscopy (JW Peng et al (2001) in Methods in Enzymology, volume 338, pp 202-230, TL James, V Dotsch, and U Schmitz, eds., Academic Press, San Diego, 2001)was employed.

[0355]¹H NMR binding experiments were performed at 25° C. in bufferedD₂O, with calcineurin concentration in the sample 0-20 μM and testedcompound concentration 0-20 μM. T2-filtered ¹H NMR spectra were obtainedon a Bruker Avance 600 instrument with CryoPhobe using low-powerpresaturation of residual water. Preacquisition delays included up tofive spin echos to suppress fast-relaxing broad protein resonances andwere times to yield in-phase signals for J-coupled spins. Chemicalshifts were referenced to internal standard 5 μM2,2-dimethylsilapentane-5-sulfonic acid (DSS).

[0356] Portions of the T2-filtered ¹H NMR spectra of 10 μM INCA1, INCA2,and INCA6 alone are displayed in the upper trace of each panel in FIGS.10A, 10B, and 10C, respectively. The INCA1 resonance labelled “a” inFIG. 10A corresponds to the R1 proton and resonances labelled “b,” “c,”and “d,” correspond to protons at the ortho, para, amd meta positions ofR2, with the R1 and R2 substituents designated as in Example 18. TheINCA2 resonances from the naphthoquinonimine core (a and c) and from thebenzenesulfonamide substituent (b, d, and e) are shown in FIG. 10B. TheINCA6 resonances from the benzene rings are shown in FIG. 1C. Acorresponding portion of the NMR spectrum taken in the presence of 20 μMcalcineurin is displayed in the lower trace of each panel. Addition ofcalcineurin caused the resonances from aromatic protons of INCA1 and thesignal from the six methyl protons, which is at a chemical shift of 2.26ppm and therefore is not shown in this segment of the spectrum, todisappear. Likewise, addition of calcineurin caused the resonancesarising from the four aromatic protons of the naphthoquinone of INCA2and the resonances from aromatic protons of INCA6 to disappear. Themethyl resonance of DSS, which was present in all experiments, was notaffected in any way by the presence of calcineurin, and was used toscale spectra for comparison. Loss of the signals on addition ofcalcineurin can be attributed to additional relaxation processes thatbecome available on binding to the protein, and indicates directinteraction of INCA compounds with calcineurin.

[0357] In NMR titration spectroscopy, saturable binding is confirmed bythe concentration dependence of the effect on resonances. In anotherseries of experiments with calcineurin and INCA compounds, the peakheight dimunition was titratable by increasing calcineurin concentrationat a single concentration of INCA compound, leading to a diminution inthe signal that was linearly proportional to the calcineurinconcentration up to a stoichiometric addition of calcineurin. A linearrelation, rather than a conventional sigmoidal binding curve, isexpected in this case, given that the estimated K_(d) values of theinteraction are at least an order of magnitude below the 10-20 μM INCAcompound concentration utilized in these NMR experiments.

[0358] These results showed that the inhibitors INCA1, INCA2, and INCA6,which displace VIVIT peptide from calcineurin, have a direct molecularinteraction with calcineurin in the absence of VIVIT peptide.

Example 20 INCA2 Selectively Inhibits NFAT Dephosphorylation In Vitro

[0359] This example illustrates that INCA2 selectively inhibits thedephosphorylation of NFAT by recombinant calcineurin in vitro.

[0360] A method for studying dephosphorylation of NFAT in vitro usesNFAT that has been phosphorylated in mammalian cells, since recombinantNFAT isolated from bacteria is not phosphorylated, and it has not beendemonstrated that NFAT can be stoichiometrically phosphorylated on thecorrect sites by protein kinases in vitro. NFAT1 is present in itsphosphorylated form in cell lysates from unstimulated T cells, and canbe detected by SDS-polyacrylamide gel electrophoresis and Westernblotting using an antiserum directed to an NFAT1 peptide (anti-67.1; AHo et al (1994) J Biol Chem 269, 28181-28186). Dephosphorylationproduces a change in mobility that is visible as a shift in theimmunostained NFAT band.

[0361] Cell lysates were made from C1.7W2 T cells washed in PBS/2 mMEGTA, 10 mM iodoacetamide, collected by centrifugation, and resuspendedat 80×10⁶ cells/ml in chilled lysis buffer consisting of 10 mM KAc, 2 mMMgAc₂, 2 mM EGTA, 100 mM HEPES/NaHEPES pH 7.4, 0.2% NP-40, 10 mMiodoacetamide, 2 mM PMSF, 20 μg/ml aprotinin, and 50 μM leupeptin. Afterincubation for 10 minutes on wet ice, the nuclei and cell debris werepelleted in a refrigerated microcentrifuge at 4° C. by centrifugationfor 5 minutes at 13,500 rpm, and the resulting supernatant recoveredwith a prechilled pipette tip, rapidly frozen using a dry ice/ethanolbath, and stored at −80° C.

[0362] For the in vitro dephosphorylation incubation, 500 nM recombinanthuman calcineurin (A and B chains; A Mondragon et al (1997) Biochemistry36, 4934-4942) and 600 nM calmodulin (Sigma) was preincubated for 15 minon ice with varied concentrations of INCA2, to allow binding ofinhibitor to equilibrate, in a reaction buffer consisting of 150 mMNaCl, 100 mM HEPES pH 7.4, 2 mM MgAc₂, 1 mM CaCl₂, 1 mg/ml BGG, 10 mMdithiothreitol, 2 mM PMSF, 20 μg/ml aprotinin, and 50 μM leupeptin.Control reactions contained calcineurin alone, or calcineurin with DMSOat a concentration comparable to that resulting from dilution of theINCA2 stock. The dephosphorylation reaction was initiated by addition of0.6 μl lysate per 20 μl reaction. After 30 minutes at 30° C., thereaction was stopped by adding 5× Laemmli buffer containing sodiumpyrophosphate and EDTA at final concentrations 20 mM and 3 mMrespectively. Samples were analyzed by SDS-polyacrylamide gelelectrophoresis according to Laemmli (U K Laemmli (1970) Nature 227,680-685) using a 6% separating gel, transferred to nitrocellulosemembrane, and probed with an antiserum to NFAT1.

[0363] The results are presented in FIG. 1A. FIGS. 11A-11B illustrategenerally that INCA2 blocks dephosphorylation of NFAT by calcineurin andthat the indicated INCA compounds (in FIG. 11B) do not blockdephoshorylation of RII phosphopeptide. In FIG. 11A, the unincubatedlysate sample (lane 1) represents the position of phosphorylated NFAT.There was no dephosphorylation when incubation was carried out in thepresence of the phosphatase inhibitor sodium pyrophosphate (lanes 3 and8). Calcineurin in the absence of inhibitors produced a cleardephosphorylation, evidenced by a shift in the NFAT band (lanes 4 and7). INCA2 at micromolar concentrations (lanes 5 and 6) blockeddephosphorylation of NFAT as effectively as VIVIT peptide (lane 2).There was a slight apparent dephosphorylation of NFAT in both the VIVITand INCA2 samples in the experiment shown, compared with lanesrepresenting unincubated lysate or lysate incubated in the presence ofthe general phosphatase inhibitor sodium pyrophosphate, reflecting theuse of a relatively high concentration of recombinant calcineurin, whichappears to dephosphorylate sites in NFAT outside the N-terminalregulatory region. The similar slight dephosphorylation in the sampleincubated with VIVIT peptide shows, however, that dephosphorylation ofthese sites does not depend on recognition of the SPRIEITPS docking siteon NFAT.

[0364] To determine whether INCA inhibitors caused a general inhibitionof calcineurin enzyme activity, in vitro assays were performed measuringthe release of free inorganic phosphate from the standard calcineurinsubstrate RII phosphopeptide. The phosphatase activity ofcalmodulin-activated calcineurin was assayed photometrically usingphosphorylated RII peptide as substrate and Malachite green fordetection (BIOMOL Research Laboratories). Assays were carried outaccording to instructions provided by the supplier of the kit, in96-well plates for 15 min at 25° C., with 6.8 nM calcineurin, 250 nMcalmodulin, and 150 μM phosphorylated RII peptide, without furtheradditions or in the presence of 100 μM INCA compound, 100 μM VIVITpeptide, or 1 μM cyclosporin A/cyclophilin A complex. DMSO concentrationwas kept constant in all samples. Release of free phosphate, calibratedto known standards, was 12-18 pmol/min under the conditions describedand was not affected by the presence of dimethyl sulfoxide (DMSO), VIVITpeptide, INCA2, or any of the other 10 INCA compounds described inExample 18. The data are plotted as a bar graph in FIG. 11B, which alsoincludes data for two of the negative control compounds from thechemical library (NEG-1 and NEG-2). Under the same conditionscyclosporin A/cyclophilin A (CsA/CypA) complex completely blockedcalcineurin enzyme activity, demonstrating that the assay would report atrue calcineurin inhibitor.

[0365] These results showed that INCA2, like the SPRIEITPS and VIVITpeptides, inhibits dephosphorylation of NFAT by calcineurin, but doesnot directly inhibit the phosphatase activity of the enzyme.

Example 21 INCA6 Inhibits Calcineurin-Dependent NFAT Activation in TCells

[0366] This example illustrates that INCA6 inhibits the activation ofNFAT elicited in T cells by treatment with the calcium ionophoreionomycin.

[0367] Dephosphorylation and nuclear import of NFAT are the earliestexperimental indicators of the physiological activation of NFAT instimulated cells (KT-Y Shaw et al (1995) Proc Natl Acad Sci USA 92,11205-11209). These indicators were assessed by SDS-polyacrylamide gelelectrophoresis and by immunocytochemistry in control and INCA6-treatedT cells. It is confirmed here that these early steps in activation areblocked by the calcineurin inhibitors cyclosporin A and FK506.

[0368] For the dephosphorylation assay, C1.7W2 T cells were pretreatedin Dulbecco's modified Eagle medium, supplemented with 10% fetal calfserum, 10 mM HEPES, and 2 mM glutamine, at 37 C. with INCA6 (5-40 μM)for 10 min, then stimulated for an additional 15 min with 1 μM ionomycinin the continued presence of inhibitor. Control incubations omittedINCA6, ionomycin, or both. DMSO was present at equal concentrations inall incubations. Cells were collected by centrifugation and lysed inLaemmli sample buffer. Samples were analyzed by SDS-polyacrylamide gelelectrophoresis according to Laemmli (U K Laemmli (1970) Nature 227,680-685) using a 6% separating gel, transferred to nitrocellulosemembrane, and probed with an antiserum to NFAT1.

[0369] The results are presented in FIG. 12A. Dephosphorylation of NFATis evident in the shift of the stained NFAT band the sample from cellsthat were stimulated with ionomycin in the absence of inhibitor (FIG.12A, lane 2). Dependence of the dephosphorylation on calcineurin isdemonstrated by blockade of the shift when cells were pretreated withCsA/FK506 (1 μM/100 nM) (not shown). Cells pretreated with INCA6 show aconcentration-dependent blockade that is partial with 10 μM INCA6 (lane5), nearly complete with 20 μM INCA6 (lane 4), and total with 40 μMINCA6 (lane 3). The inhibitor does not produce a general impairment ofintracellular signalling, since activation of the PMA-MAP kinasesignalling pathway was not blocked by INCA6 under these conditions.Activation of the latter pathway was monitored by examiningphosphorylation of p44/p42 MAP kinase. For this assay the cells werepretreated and incubated as for the NFAT dephosphorylation assay, exceptthat stimulation was for 15 min with 20 nM PMA. Cells were collected bycentrifugation, lysed in Laemmli sample buffer, and analyzed by Westernblotting with an anti-phospho-p44/p42 MAP kinase antibody (CellSignaling Technology). The results of the MAP kinase phosphorylationassay are presented in FIG. 12B.

[0370] For an immune staining assay, C1.7W2 T cells growing oncoverslips in Dulbecco's modified Eagle medium, supplemented with 10%fetal calf serum, 10 mM HEPES, and 2 mM glutamine, at 37 C. werepretreated with medium alone, with INCA6 (20 μM or 40 μM), or withCsA/FK506 (1 μM/100 nM) for 10 min, then stimulated for an additional 15min with 1 μM ionomycin, in medium alone or in the continued presence ofinhibitor as appropriate. Medium was removed, and the cells were fixed,permeabilized, and stained using the anti-67.1 antiserum against NFAT1(A Ho et al (1994) J Biol Chem 269, 28181-28186) and a fluorescentsecond antibody. The results are presented in FIG. 13. The fluorescencemicroscopy micrographs presented in FIG. 13 demonstrate that NFAT immunestaining was localized to the cytoplasm in unstimulated cells, localizedto the nucleus in cells stimulated with ionomycin in the absence ofinhibitor, but localized to the cytoplasm in cells stimulated withionomycin in the presence of either concentration of INCA6 or in thepresence of cyclosporin A/FK506 (CsA/FK506).

[0371] These results showed that exposure of C1.7W2 T cells to INCA6prevented the early changes that are hallmarks of calcineurin-dependentactivation of NFAT in stimulated cells.

Example 22 INCA6 Inhibits Induction of Cytokine mRNAs that are Targetsof NFAT in T Cells

[0372] This example illustrates that, under stimulation conditions thatmimic physiological stimulation of T cells, INCA6 is as effective asCsA/FK506 in inhibiting the early induction of several cytokine mRNAsthat are the targets of calcineurin-NFAT signalling.

[0373] The effects of INCA6 on transcription were examined in the caseof several cytokine mRNAs that are known targets of calcineurin-NFATsignalling. C1.7W2 T cells were preincubated for 10 min. in mediumalone, in medium containing INCA6, or in medium containing CsA andFK506, then stimulated under conditions that normally initiatesignalling through the calcium-calmodulin-calcineurin-NFAT pathway.Cells in control samples that had not been pretreated with inhibitorwere left unstimulated, or were stimulated for 45 min with 20 nM PMAalone, or with 20 nM PMA and 1 μM ionomycin. Cells in parallel samplesthat had been pretreated for 10 min with inhibitor were then furtherincubated for 45 min without stimulation or with PMA and ionomycin, inthe continued presence of inhibitor. At the end of the incubation, cellswere harvested and total cellular RNA was extracted using UltraspecRNA-binding resin (Biotecx Laboratories). Levels of cytokine mRNAs wereanalyzed by RNase protection assays performed with RiboQuant multiprobekits (Pharmingen) following the instructions of the manufacturer.Briefly, for each sample 10 μg of target RNA was hybridized overnight toa ³²P-labelled RNA probe that had been synthesized in vitro from atemplate set representing multiple murine cytokine mRNAs, and then theunhybridized probe and other single-stranded RNAs were digested withRNases. The protected RNAs were purified, and probes for the individualcytokine mRNAs were resolved on a denaturing 6% polyacrylamide gel.Transcript levels were quantified by autoradiography and by use of aphosphorimager (Molecular Dynamics). Probes for the individual cytokinetranscripts were identified by the length of the respective protectedfragments. The results are presented in FIGS. 14A to 14C, which arepictures of RNA gel autoradiograms. Data were corrected for minorvariations in RNA loading by normalizing the data for each sample to theintensity of the signal for the housekeeping transcripts L32 and GAPDH(see lower panels of FIGS. 14A, 14B, and 14C).

[0374] FIGS. 14A-14C are pictures of autoradiograms of RNA gelsillustrating that INCA6 blocks the NFAT-dependent expression ofendogenous cytokine genes. FIGS. 14A-14C show protected segments ofcomplementary RNA probes, which indicate the presence and level ofspecific cytokine mRNAs in the corresponding samples. As illustrated inFIG. 14A, GM-CSF mRNA was detectable in unstimulated cells, and itslevel was unchanged in cells stimulated with PMA alone. The level ofGM-CSF mRNA was sharply increased by treatment with PMA and ionomycin.The basal level of GM-CSF mRNA was not affected by treatment with INCA6or with CsA/FK506 (1 μM/100 nM), but the induction was partiallyprevented by 20 μM INCA6, and fully blocked by 40 μM INCA6 or byCsA/FK506. M-CSF mRNA showed only a modest induction over its basallevel on treatment for 45 min with PMA and ionomycin, and so was notfurther analyzed. The mRNAs for the housekeeping genes L32 and GAPDH arepresent at similar levels in all lanes, indicating little variation inmRNA loading on the gel.

[0375]FIG. 14B shows that there was a slight elevation of TNFα mRNAlevel on stimulation with PMA alone, and a more pronounced induction onstimulation with PMA and ionomycin. The induction of TNFα mRNA wasinhibited by 20 μM or 40 μM INCA6 and by CsA/FK506. IFNγ mRNA wasinduced only in cells stimulated with the combination of PMA andionomycin. Here again, induction was inhibited by INCA6 and byCsA/FK506. The level of TNFβ mRNA was unaltered by any condition tested.

[0376]FIG. 14C documents that lymphotactin (Ltn), MIP1β, and MIP-1αmRNAs were induced in response to stimulation with PMA and ionomycin,that induction was partially blocked by 20 μM INCA6, and that inductionwas fully blocked by 40 μM INCA6 or by CsA/FK506. RANTES mRNA andinterferon γ-inducible protein of 10 kDa (IP-10) mRNA served as controlsthat were unaffected by the stimulation conditions assayed and by theinhibitors.

[0377] Recapitulating, treatment of C1.7W2 T cells with PMA andionomycin caused rapid induction of the mRNAs for GM-CSF, IFNγ, TNFα,lymphotactin, MIP1α and MIP1β. Consistent with previous work, theincrease in levels of these mRNAs was blocked by inhibiting calcineurinwith CsA/FK506. Cytokine mRNA induction was likewise inhibited by 20 μMor 40 μM INCA6, with 40 μM INCA6 reducing mRNA levels to those ofunstimulated cells. Levels of TNFβ, RANTES, and IP-10 mRNAs, which arenot downstream targets of NFAT, were not increased at early times bytreatment with PMA and ionomycin and were unaffected by INCA6.

[0378] These results showed that exposure of C1.7W2 T cells to 1NCA6prevented the induced transcription of cytokine genes that aredownstream targets of NFAT.

Example 23 Certain Structural Analogues of INCA1, INCA2 and INCA6 areInhibitors of the Calcineurin-VIVIT Peptide Interaction

[0379] This example illustrates that the three inhibitors INCA1, INCA2,and INCA6 are representative of three families of organic compounds thatinterfere with the calcineurin-VIVIT interaction.

[0380] The ability of commercially available structural analogues ofINCA1, INCA2, and INCA6 to inhibit calcineurin-VIVIT interaction wasevaluated using the fluorescence polarization assay of Example 18. Eachcompound was incubated, at a range of concentrations, with calcineurinand OG-VIVIT, and the ability of the tested compound to displace boundpeptide from calcineurin was determined by measuring the polarization ofOG-VIVIT fluorescence. FIGS. 15A-15C illustrate three general chemicalstructures of inhibitors of the protein-protein interaction betweencalcineurin and NFAT, typical chemical modifications that can be madethereto, and the inhibitory activity exhibited by such modifiedcompounds. The general structures of INCA1 (FIG. 15A), INCA2 (FIG. 15B),and INCA6 (FIG. 15C) are presented, and the modifications to each Rgroup for each analog are indicated. In FIG. 15B, the dashed line in thering system represents a single bond for compound 2D and a double bondfor other compounds; the dashed line between the nitrogen atom and thering bearing substituents R¹-R³ represents a single bond for compounds2H, 2I, and 2K, and a double bond for other compounds. In FIG. 15C, thedashed lines represent single bonds for compounds 6A, 6B, and 6D, anddouble bonds for other compounds. Ac=acetyl;DDC=4,4-dimethyl-2,6-dioxocyclohexyl; Et=ethyl; Me=methyl; andPh=phenyl. K_(i) values were estimated as described in Example 18 forthose compounds that showed inhibitory activity and are indicated in thelast column of FIGS. 15A-15C.

[0381] In many cases the inhibitory potency was only marginally affectedby conservative changes in ring substituents. However, certain changescaused moderate to dramatic losses of potency. For example, reduction ofthe vicinal keto groups of INCA1 to hydroxyl groups, or theirreplacement by halogen substituents, resulted in inactive compounds.Likewise, introduction of bulky substituents at R¹ in INCA2, orreduction of INCA6 to the hydroquinone or methoxyquinone, caused apronounced decrease in or a loss of inhibitory activity. Thesedistinctive structure-activity profiles are the signature of a specificligand-protein interaction.

[0382] These results demonstrate that structural modifications to INCAcompounds result in additional organic compounds that could be useful ininhibiting calcineurin-NFAT signalling.

Example 24 Gene Targets of NFAT and Identification of Same

[0383] Transcriptional targets of the NFAT proteins vary depending onthe cellular context, including signals impinging on the cell, thestructure of cellular chromatin, and the presence in the cell of otherconstitutively active or induced transcription factors. In a givencellular context, NFAT induces expression of a specific panel of genesrelated to cell differentiation or cell function. NFAT works in concertwith other transcription factors, and it is generally observed thatblockade of NFAT activation or ablation of its binding site(s) in DNAcan reduce or eliminate target gene expression.

[0384] Familiar target genes of NFAT are cytokine and activation genesin the immune system. Cell surface receptors that activate immune cells,e.g. the T cell receptor, the macrophage Fcγ receptor, or the mast cellFcε receptor, signal through NFAT to induce expression of cytokinegenes, e.g. the IL2, TNFα, IL4, IL5, IL13, and GM-CSF genes, dependingon cellular context, and a variety of other activation genes, some ofwhich are listed herein. Immunosuppressants CsA and FK506 has been usedto block induction of this array of genes by inhibiting signalling inthe calcineurin-NFAT pathway.

[0385] Under other conditions, NFAT has a central role in initiatingimmune tolerance or anergy in T cells and B cells. Due to the cellularcontext and the presence or absence of specific environmental inputs,NFAT signalling is addressed in this case to a different set of targetgenes, including diacylglycerol kinase-α, Itch, Cbl-b, and Jumonji.

[0386] Yet another set of targets is controlled by NFAT in the heart,when physiological stresses lead to initiation of pathological cardiachypertrophy. Genes known to be targets of NFAT in this process are theatrial natriuretic factor (ANF), B-type natriuretic peptide (BNP), andadenylosuccinate synthetase-1 genes. Given the critical role that NFATproteins have been shown to play in initiation and development ofcardiac hypertrophy, it is unlikely that its direct targets are limitedto these few indicator genes. A skilled practitioner will be able todetermine, through methods known in the art, which of the many othergenes induced during myocardial hypertrophy are direct NFAT targets.

[0387] NFAT proteins also participate in osteoclast differentiation andbone resorption. The osteoclast markers TRAP and calcitonin receptor areNFAT target genes and, again, routine experimentation will show whichother genes expressed by osteoclasts are direct NFAT targets.

[0388] NFAT targets also include binding sites that support reactivationof latent viruses and viral growth, for example the reactivation oflatent Kaposi's sarcoma-associated herpesvirus (KSHV; also termed humanherpesvirus-8) and the replication of HIV.

[0389] Among other NFAT targets are genes that have functions inphysiological and pathological processes in a variety of tissues, forexample the COX-2 and iNOS genes. Biological processes where regulationof these genes by NFAT is implicated include inflammation, angiogenesis,and tumor invasion.

[0390] Whether any specific gene or set of genes is an NFAT target canbe determined using methods known to those of ordinary skill in the art.Several such methods are described below.

[0391] In one method, a constitutively active NFAT protein is expressedin cells and transcription of the gene is assayed by RT-PCR to determinewhether or not it is influenced by NFAT expression (see, e.g., Macian etal., Cell 109: 719-731 (2002)).

[0392] In a another method, cells are transfected or infected to expresshigh levels of GFP-VIVIT, then stimulated to induce an increase inintracellular calcium levels, with or without activation of otherintracellular signalling pathways. Under these conditions GFP-VIVIT canblock the induced transcription of genes that are direct or indirecttargets of NFAT (see, e.g., Aramburu et al., Science 285: 2129-2133(1999)).

[0393] In another method, gene expression is evaluated by RT-PCR or DNAarray analysis in cells lacking one or more members of the NFAT family,and compared to expression in wildtype cells expressing all NFAT familymembers (see, e.g., Macian et al., Cell 109: 719-731 (2002)).

[0394] In another method, genes that are potentially direct targets ofNFAT are identified by using bioinformatic techniques to find noncoding(putative regulatory) sequences that are highly conserved across species(see, e.g., Loots et al., Genome Res. 12: 832-839 (2002). These regionsmay then be examined for the presence of NFAT:AP-1, NFAT-dimer, or highaffinity NFAT binding sites according to criteria known in the art (see,e.g., Kel et al., J Mol Biol. 288: 353-376 (1999) and Rao et al., AnnualReview of Immunology 15: 707-747 (1997)) or derived from knownNFAT-binding sequences. Candidate NFAT target sites can then be analyzedexperimentally.

[0395] In another method, regions within a gene that contain regulatorysequences can be identified experimentally by analyzing the gene locusfor the presence of inducible DNase I hypersensitive sites (see, e.g.,Cockerill, Methods Mol Biol. 130: 29-46 (2000), Carey et al.,Transcriptional regulation in eukaryotes: Concepts, strategies andtechniques. Chapter 10, In vivo analysis of an endogenous controlregion. Cold Spring Harbour Laboraotry Press (2000), Agarwal et al.,Immunity 9: 765-775 (1998), and Agarwal et al., Immunity 12:643-652(2000)), and candidate NFAT sites in these regions can be analyzedexperimentally.

[0396] In another method, the proximal promoter of the gene, or aputative regulatory region found by either of the above two techniques,is incorporated into a reporter plasmid, the plasmid is transfected intocells with or without co-expressed NFAT, the cells are stimulatedappropriately to activate NFAT, and reporter activity is assessed. Ifthe regulatory region contains functional NFAT binding sites, reporteractivity will be induced under the same stimulation conditions thatactivate NFAT; reporter activity will be influenced by expression ofexogenous NFAT or constitutively-active NFAT; and mutation ofNFAT-binding sites in the regulatory region will abrogate induction ofreporter activity (see, e.g., Carey et al., Transcriptional regulationin eukaryotes: Concepts, strategies and techniques, Chapter 5,Functional assays for promoter analysis. Cold Spring Harbour LaboraotryPress (2000))

[0397] Another method involves determining whether the promoter or otherregulatory regions of the gene are represented in a chromatinimmunoprecipitation assay, in which NFAT proteins and other DNA-bindingproteins are crosslinked to the genomic regions to which they are boundin living cells, following which immunoprecipitation is performed usingspecific antibodies to NFAT, and the presence in the immunoprecipitateof the genomic sequences of interest is determined by PCR (see, e.g.,Avni et al., Nature Immunol 3: 643-651(2002).

[0398] Those skilled in the art will be able to ascertain using no morethan routine experimentation, many equivalents of the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. A pharmaceutical composition comprising atherapeutically effective amount of an organic molecule capable ofinhibiting protein-protein interaction between calcineurin and NFAT, anda pharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1, wherein the agent inhibits dephosphorylation of NFAT bycalcineurin.
 3. The pharmaceutical composition of claim 1, wherein themolecular weight of the organic molecule is less than 2500 Da.
 4. Thepharmaceutical composition of claim 1, wherein the molecular weight ofthe organic molecule is between about 100 and 2000 Da.
 5. Thepharmaceutical composition of claim 1, wherein the molecular weight ofthe organic molecule is between about 200 and 1500 Da.
 6. Thepharmaceutical composition of claim 1, wherein the molecular weight ofthe organic molecule is between about 300 and 1000 Da.
 7. Thepharmaceutical composition of claim 1, wherein the organic moleculebinds calcineurin with an affinity constant of at least about 2×10⁴ M⁻¹.8. The pharmaceutical composition of claim 1, wherein the organicmolecule binds calcineurin with an affinity constant of at least about10⁶ M⁻¹.
 9. The pharmaceutical composition of claim 1, wherein theorganic molecule binds calcineurin with an affinity constant of at leastabout 10⁷ M⁻¹.
 10. The pharmaceutical composition of claim 1, whereinthe organic molecule binds calcineurin with an affinity constant of atleast about 10⁸ M⁻¹.
 11. The pharmaceutical composition of claim 1,wherein the organic molecule is a compound selected from the groupconsisting of: formula (I):

wherein: R¹ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionallysubstituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3R⁶; heteroaryl optionally substituted with 1-4 R⁶; C₂-C₈ alkenyl, orC₂-C₈ alkynyl, cyano, nitro, carboxy, carbo(C₁-C₆)alkoxy, trihalomethyl,halogen, C₁-C₆ alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl,arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, or ureido; R² is C₁-C₂₀ alkyl optionallysubstituted with 1-20 R₆, C₃-C₈ cycloalkyl optionally substituted with1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionallysubstituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶,C₁-C₆ alkoxy, or hydroxy; R³ is hydrogen or halogen; R⁴ is hydrogen,C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyloptionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4R⁶, heterocyclyl optionally substituted with 1-3 R⁶, heteroaryloptionally substituted with 1-4 R⁶, or halogen; R is NR⁷, O or S; R⁶ ishalogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl,alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino,diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl,alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl,alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl,acyloxy, cyano, mercapto or ureido; and R⁷ is C1-C6 alkyl; formula (II):

wherein: R¹ and R² are each independently hydrogen, halogen, amino,C₁-C₆alkylamino, di(C₁-C₆)alkylamino, arylamino, diarylamino, or4,4-dimethyl-2,6-dioxocyclohexyl; R³ is NR¹¹ or O; R⁴, R⁵ and R⁸ areeach independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro,haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino,dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;R⁶ is hydrogen, halogen, or when taken together with R⁷ forms a doublebond between the carbon atoms to which they are attached; R⁷ ishydrogen, halogen, or when taken together with R⁶ forms a double bondbetween the carbon atoms to which they are attached; R⁹ is OR¹³, or whentaken together with R¹⁰ forms a double bond between the carbon andnitrogen atoms to which they are attached; R¹⁰ is hydrogen, or whentaken together with R⁹ forms a double bond between the carbon andnitrogen atoms to which they are attached; R¹¹ is SO₂R¹²; and R¹² isaryl optionally substituted with alkyl; R¹³ is alkyl or aryl; andformula (III):

wherein, R¹ and R⁴ are each independently O or NR⁸; R² and R³ are eachindependently hydrogen, halogen, or R² and R³ together combine to formaryl optionally substituted with 1-4 R⁹; R⁵ is hydrogen, halogen,carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, orcyano; R⁶, R⁷ and R⁹ are each independently hydrogen, C₁-C₆ alkyl,halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy,aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino,acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano,mercapto or ureido; R⁸ is SO₂R¹⁰; and R¹⁰ is aryl optionally substitutedwith alkyl.
 12. A method for inhibiting protein-protein interactionbetween calcineurin and NFAT, comprising: providing calcineurin andNFAT; providing the pharmaceutical composition of claim 1; andcontacting the calcineurin, NFAT, and pharmaceutical compositiontogether, such that the protein-protein interaction between calcineurinand NFAT is inhibited.
 13. A method of inhibiting an immune response inan animal, comprising administering to the animal the pharmaceuticalcomposition of claim
 1. 14. A method for treating a disease or conditioninvolving hyperactivity or inappropriate activity of the immune system,comprising: identifying an animal suffering from a disease or conditioninvolving hyperactivity or inappropriate activity of the immune system;and administering to the animal a therapeutically effective amount ofthe pharmaceutical composition of claim 1, to thereby treat the diseaseor condition involving hyperactivity or inappropriate activity of theimmune system.
 15. The method of claim 14, wherein the organic moleculeis a compound selected from the group consisting of: formula (I):

wherein: R¹ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionallysubstituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3R⁶; heteroaryl optionally substituted with 1-4 R⁶; C₂-C₈ alkenyl, orC₂-C₈ alkynyl, cyano, nitro, carboxy, carbo(C₁-C₆)alkoxy, trihalomethyl,halogen, C₁-C₆ alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl,arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, or ureido; R² is C₁-C₂₀ alkyl optionallysubstituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionallysubstituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶,C₁-C₆ alkoxy, hydroxy; R³ is hydrogen or halogen; R⁴ is hydrogen, C₁-C₂₀alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionallysubstituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶,heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionallysubstituted with 1-4 R⁶, or halogen; R⁵ is NR⁷, O or S; R⁶ is halogen,hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino,acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano,mercapto or ureido; and R⁷ is C1-C6 alkyl; formula (II):

wherein: R¹ and R² are each independently hydrogen, halogen, amino,C₁-C₆alkylamino, di(C₁-C₆)alkylamino, arylamino, diarylamino, or4,4-dimethyl-2,6-dioxocyclohexyl; R³ is NR¹¹ or O; R⁴, R⁵ and R⁸ areeach independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro,haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino,dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;R⁶ is hydrogen, halogen, or when taken together with R⁷ forms a doublebond between the carbon atoms to which they are attached; R⁷ ishydrogen, halogen, or when taken together with R⁶ forms a double bondbetween the carbon atoms to which they are attached; R⁹ is OR¹³, or whentaken together with R¹⁰ forms a double bond between the carbon andnitrogen atoms to which they are attached; R¹⁰ is hydrogen, or whentaken together with R⁹ forms a double bond between the carbon andnitrogen atoms to which they are attached; R¹¹ is SO₂R¹²; and R¹² isaryl optionally substituted with alkyl; R¹³ is alkyl or aryl; andformula (III):

wherein, R¹ and R⁴ are each independently O or NR⁸; R² and R³ are eachindependently hydrogen, halogen, or R² and R³ together combine to formaryl optionally substituted with 1-4 R⁹; R⁵ is hydrogen, halogen,carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, orcyano; R⁶, R⁷ and R⁹ are each independently hydrogen, C₁-C₆ alkyl,halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy,aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino,acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano,mercapto or ureido; R⁸ is SO₂R¹⁰; and R¹⁰ is aryl optionally substitutedwith alkyl.
 16. The method of claim 14 wherein the disease or conditioninvolving hyperactivity or inappropriate activity of the immune systemis selected from the group consisting of: an acute immune disease, achronic immune disease, and an autoimmune disease.
 17. A method fortreating a disease involving excessive or inappropriate activation ofNFAT, or a molecular target thereof, comprising: identifying an animalsuffering from a disease involving excessive or inappropriate activationof NFAT or a molecular target thereof, and administering to the animal atherapeutically effective amount of the pharmaceutical composition ofclaim 1, to thereby treat the disease involving excessive orinappropriate activation of NFAT or molecular target thereof.
 18. Aprocess of making an agent that inhibits protein-protein interactionbetween calcineurin and NFAT, the process comprising: carrying out amethod to identify an agent that inhibits protein-protein interactionbetween calcineurin and NFAT, wherein the method comprises: providing afirst compound selected from the group consisting of calcineurin or abiologically active derivative thereof, and NFAT or a biologicallyactive derivative thereof; providing a second compound selected from thegroup consisting of calcineurin or a biologically active derivativethereof, and NFAT or a biologically active derivative thereof, whereinthe second compound is different from the first compound, and whereinthe second compound is labeled; providing a candidate agent; contactingthe first compound, the second compound, and the candidate agent witheach other; and determining the amount of label bound to the firstcompound, wherein a reduction in interaction between the first compoundand the second compound as assessed by label bound is indicative ofusefulness of the candidate agent in inhibiting protein-proteininteraction between calcineurin and NFAT; and manufacturing the agent,to thereby make an agent that inhibits protein-protein interactionbetween calcineurin and NFAT.
 19. The process of claim 18, wherein thefirst compound is calcineurin and the second compound is a biologicallyactive derivative of NFAT.
 20. The process of claim 18, wherein thebiologically active derivative of NFAT comprises the amino acid sequenceGPHPVIVITGPHEE.
 21. A method of manufacturing an agent that inhibitsprotein-protein interaction between calcineurin and NFAT, the methodcomprising: providing an organic compound capable of inhibitingprotein-protein interaction between calcineurin and NFAT; providing atleast one pharmaceutically acceptable carrier; and combining the organiccompound with the pharmaceutically acceptable carrier, to therebymanufacture an agent that inhibits protein-protein interaction betweencalcineurin and NFAT.
 22. The method of claim 21, further comprising thestep of manufacturing the agent into a form suitable for administrationto an animal via a route selected from a group consisting of: oral,parenteral, topical, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intrasternal.
 23. A method for inhibiting protein-protein interactionbetween calcineurin and NFAT, comprising: providing calcineurin andNFAT; providing an organic molecule capable of inhibitingprotein-protein interaction between calcineurin and NFAT, wherein theorganic molecule is a compound selected from the group consisting of:formula (I):

wherein: R¹ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionallysubstituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3R⁶; heteroaryl optionally substituted with 1-4 R⁶; C₂-C₈ alkenyl, orC₂-C₈ alkynyl, cyano, nitro, carboxy, carbo(C₁-C₆)alkoxy, trihalomethyl,halogen, C₁-C₆ alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl,arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, or ureido; R² is C₁-C₂₀ alkyl optionallysubstituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionallysubstituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶,C₁-C₆ alkoxy, hydroxy; R³ is hydrogen or halogen; R⁴ is hydrogen, C₁-C₂₀alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionallysubstituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶,heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionallysubstituted with 1-4 R⁶, or halogen; R⁵ is NR⁷, O or S; R⁶ is halogen,hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino,acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano,mercapto or ureido; and R⁷ is C₁-C₆ alkyl; formula (II):

wherein: R¹ and R² are each independently hydrogen, halogen, amino,C₁-C₆alkylamino, di(C₁-C₆)alkylamino, arylamino, diarylamino, or4,4-dimethyl-2,6-dioxocyclohexyl; R³ is NR¹¹ or O; R⁴, R⁵ and R⁸ areeach independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro,haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino,dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;R⁶ is hydrogen, halogen, or when taken together with R⁷ forms a doublebond between the carbon atoms to which they are attached; R⁷ ishydrogen, halogen, or when taken together with R⁶ forms a double bondbetween the carbon atoms to which they are attached; R⁹ is OR¹³, or whentaken together with R¹⁰ forms a double bond between the carbon andnitrogen atoms to which they are attached; R¹⁰ is hydrogen, or whentaken together with R⁹ forms a double bond between the carbon andnitrogen atoms to which they are attached; R¹¹ is SO₂R¹²; and R¹² isaryl optionally substituted with alkyl; R¹³ is alkyl or aryl; andformula (III):

wherein, R¹ and R⁴ are each independently O or NR⁸; R² and R³ are eachindependently hydrogen, halogen, or R² and R³ together combine to formaryl optionally substituted with 1-4 R⁹; R⁵ is hydrogen, halogen,carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, orcyano; R⁶, R⁷ and R⁹ are each independently hydrogen, C₁-C₆ alkyl,halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy,aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino,acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano,mercapto or ureido; R⁸ is SO₂R¹⁰; and R¹⁰ is aryl optionally substitutedwith alkyl; and contacting the calcineurin, NFAT, and organic moleculetogether such that protein-protein interaction between the calcineurinand the NFAT is inhibited.